Methods and devices for modulating cellular activity using ultrasound

ABSTRACT

The present invention comprises methods and devices for modulating the activity or activities of living cells, such as cells found in or derived from humans, animals, plants, insects, microorganisms and other organisms. Methods of the present invention comprise use of the application of ultrasound, such as low intensity, low frequency ultrasound, to living cells to affect the cells and modulate the cells&#39; activities. Devices of the present invention comprise one or more components for generating ultrasound waves, such as ultrasonic emitters, transducers or piezoelectric transducers, composite transducers, CMUTs, and which may be provided as single or multiple transducers or in an array configurations. The ultrasound waves may be of any shape, and may be focused or unfocused.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/192,749, filed Jun. 24, 2016, now U.S. Pat. No. 10,556,132, which isa continuation of U.S. patent application Ser. No. 14/460,007, filedAug. 14, 2014, now U.S. Pat. No. 9,403,038, which is a continuation ofU.S. patent application Ser. No. 14/025,586, filed Sep. 12, 2013, nowU.S. Pat. No. 8,858,440, which is a continuation of U.S. patentapplication Ser. No. 13/003,853, filed Apr. 6, 2011, now U.S. Pat. No.8,591,419, which is a National Stage Entry of PCT/US2009/050560, filedJul. 14, 2009, which claims priority to U.S. Provisional ApplicationSer. No. 61/080,666, filed Jul. 14, 2008, and to U.S. ProvisionalApplication Ser. No. 61/175,413, filed May 4, 2009, the full disclosuresof which are incorporated herein by reference.

FIELD

The present invention relates to ultrasound modulation of cellularactivities, including nerves and other cells found in human and animals.

BACKGROUND

Ultrasound (US) has been used for many medical applications, and isgenerally known as cyclic sound pressure with a frequency greater thanthe upper limit of human hearing. The production of ultrasound is usedin many different fields, typically to penetrate a medium and measurethe reflection signature or supply focused energy. For example, thereflection signature can reveal details about the inner structure of themedium. A well known application of this technique is its use insonography to produce a picture of a fetus in a womb. There are otherapplications which may provide therapeutic effects, such as lithotripsyfor ablation of kidney stones or high-intensity focused ultrasound forthermal ablation of brain tumors.

A benefit of ultrasound therapy is its non-invasive nature. For example,methods for modulating neural activity include both invasive andnon-invasive techniques. Neuromodulation techniques such as deep brainstimulation (DBS) and repetitive transcranial magnetic stimulation havegained attention due to their therapeutic utility in the management ofnumerous neurological/psychiatric diseases. These methods forstimulating neuronal circuits have been demonstrated to hold promise forthe treatment of such diseases and disorders as Parkinson's,Alzheimer's, coma, epilepsy, stroke, depression, schizophrenia,addiction, neurogenic pain, cognitive/memory dysfunction, and others. Inthe laboratory setting, recent work demonstrated efficacy formillisecond optical control of individual neurons and synapses in intactbrain circuits.

The current goals of neurostimulation techniques are to modulateneuronal activity and thereby nervous system function by deliveringexogenous energy to intact circuits. However, many of these techniques,such as DBS and vagus nerve stimulation (VNS) require the surgicalimplantation of stimulating electrodes, an invasive, expensive and evendangerous procedure. For example, the surgical implantation ofstimulating electrodes increases secondary medical risks such asinfection. The primary cost associated with the surgical implantation ofneurostimulation devices is approximately $17,000 to $60,000 perpatient, which costs do not take into account the significant costs ofpre- and post-operative care.

Ultrasound refers to cyclical vibrations in a frequency range abovehuman hearing, i.e., above about 20 thousand cycles per second(kilohertz, kHz) and including vibrational frequencies of tens andhundreds of millions of cycles per second (MegaHertz, MHz), e.g., arange from about 0.02 to 200 MHz. Ultrasound was first shown to becapable of modulating neuronal activity by inducing reversiblesuppression. It was earlier demonstrated that ultrasound delivered tothe lateral geniculate nucleus of cats in vivo, reversibly suppressedlight-evoked potentials in the visual cortex.

Approaches to affecting neural activity in the brain using ultrasoundhave employed ultrasound frequencies above about 0.6 MHz applied forextended periods of times (several seconds to several minutes), and atintensity levels above about 10 Watts per square centimeter (mW/cm²,where 1 mW=10⁻³ Watts, and 1 cm=10⁻² meters). Many of these approachesare intended to produce macroscopic effects, such as tissue ablationduring high intensity focused ultrasound (HIFU). Ultrasound frequenciesused for imaging typically range from 2.5 to 7.5 MHz.

What are needed are non-invasive and effective therapies for modulatingcellular activity, including the activity of neural cells and othertypes of cells.

SUMMARY OF THE DISCLOSURE

The present invention comprises methods and devices for modulating theactivity or activities of living cells, such as cells found in orderived from humans, animals, plants, insects, microorganisms and otherorganisms. Methods of the present invention comprise use of theapplication of ultrasound (US), such as low-intensity, low-frequencyultrasound, to living cells to affect the cells and modulate the cells'activities. Devices of the present invention comprise one or morecomponents for generating ultrasound waves, such as ultrasonic emitters,transducers or piezoelectric transducers, composite transducers, CMUTs(capacitive micromachined ultrasound transducers), and may be providedas single or multiple transducers or in an array configurations. Theultrasound waves may be of any shape, and may be focused or unfocused,depending on the application desired. The ultrasound may be at anintensity in a range of about 0.0001 to about 900 mW/cm² and anultrasound frequency in a range of about 0.02 to about 1.0 MHz at thesite of the tissue to be modulated.

Methods of the present invention comprise modulating cellular activityby providing ultrasound waves to cells or tissues at an effectiveintensity and for effective time range so that the cell activity isaltered. Methods comprise treatment of physiological or pathologicalconditions including, but not limited to, Parkinson's disease,Alzheimer's disease, coma, epilepsy, stroke, depression, schizophrenia,addiction, neurogenic pain, cognitive/memory dysfunction, diabetes,obesity, obsessive compulsive disorders, traumatic brain injury,post-traumatic stress disorder (PTSD), coma, minimally conscious orvegetative states, locked in syndrome, spinal cord injuries, peripheralneuropathies, migraine, epilepsy, and other pathologies associated withorgans of the human or animal body. Methods comprise mapping of thebrain, stimulating or inhibiting nerve activity such as the vagus nerve,stimulating physiological responses of cells, tissues, or organs,photoacoustic tomography, and other uses of ultrasound waves in thebody.

A method of the present invention comprises acoustically couplingcomponent for generating ultrasound waves, such as an ultrasoundtransducer, to an external surface or inside the body of an animal,human, insect, plant, or to plates or containers of cells or tissues.The ultrasound transducer is driven to form in the cells, tissues, ororgans pressure fluctuations, a stimulus waveform, with an intensityabove about 0.001 milliWatts per square centimeter (mW/cm²) and belowabout 900 mW/cm² and an ultrasound frequency below about 1.0 MegaHertz(MHz), from about 0.02 MHz to about 1.0 MHz, at the site of the tissueto be manipulated.

A method of the present invention comprises treating disorderscomprising acoustically coupling an ultrasound transducer to an externalsurface of a subject or container to be treated. The ultrasoundtransducer is driven to deliver an effective dose of ultrasound at anintensity above about 20 mW/cm² and below about 900 mW/cm² and anultrasound frequency below about 1.0 MHz at the site of the tissue orcells to be manipulated.

A device of the present invention may comprise logic encoded in tangibleform that is configured to perform one or more steps of the abovemethods.

DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIG. 1 shows a block diagram that illustrates an example system formodulating neural activity.

FIG. 2 shows a graph that illustrates an example ultrasound waveform formodulating neural activity.

FIG. 3 shows a flow diagram that illustrates, at a high level, a methodfor modulating neural activity.

FIG. 4A shows an illustration of an experimental setup to demonstrateeffects on neural activity from an ultrasound waveform.

FIG. 4B shows a graph that illustrates an example acoustic signalreceived at a location of neural tissue.

FIG. 4C shows a graph that illustrates an example spectrum of theacoustic signal depicted in FIG. 4B.

FIG. 5 shows a graph that illustrates example temporal response ofneural activity after modulation.

FIG. 6A shows a graph that illustrates comparative temporal responses ofneural activity after modulation by electrical impulses and aftermodulation by an ultrasound waveform.

FIG. 6B shows a graph that illustrates temporal electrical responses ofa neuron after modulation by an ultrasound waveform.

FIG. 7 shows a graph that illustrates example effects of some processinhibitors on neural activity modulated by an ultrasound waveform,according to an embodiment; is an image that illustrates an exampleeffect on neural sodium (Na⁺) transients after modulation by anultrasound waveform.

FIG. 8 shows a graph that illustrates an example temporal effect onneural sodium (Na⁺) transients after modulation by an ultrasoundwaveform.

FIG. 9 shows a graph that illustrates example temporal effects on neuraland glial calcium (Ca²⁺) transients after modulation by an ultrasoundwaveform.

FIG. 10 shows a graph that illustrates an example temporal effect onneural presynaptic activity by modulation with an ultrasound waveform.

FIG. 11 shows a graph that illustrates example enhanced effects onneural activity by modulation with particular ultrasound waveforms.

FIG. 12 shows a block diagram that illustrates a computer system uponwhich an embodiment of the invention may be implemented.

FIG. 13 shows a drawing of methods of the present invention forstimulating the vagus nerve efferents to stimulate insulin synthesisand/or pancreatic secretions and/or activating β cells or other cells ofthe pancreas.

FIG. 14 shows a drawing of methods of the present invention for directstimulation of the pancreas and its cells to stimulate insulin synthesisand/or pancreatic secretions and/or activating β cells or other cells ofthe pancreas. Such a method may be used alone or in conjunction withstimulation of the vagal nerve.

FIG. 15 shows a drawing of methods of the present invention forproviding a sweeping ultrasound field to deliver unfocused waves oflow-intensity ultrasound to multiple gastrointestinal regions.

FIG. 16 shows a drawing of methods of the present invention for an arrayof transducer components to affect vagus nerve efferent and afferentactivity for treatment of obesity.

FIG. 17 shows drawings of methods of the present invention for use of anarray of transducers for treatment of head injuries that can be portableor used on site by first responders or emergency room personnel.

FIG. 18A shows the illustration of the method used to transmit laterallyfocused ultrasound stimulus waveforms to intact motor cortex.

FIG. 18B shows an example of the strategy and parameters used inconstructing low-intensity US stimulation waveforms.

FIG. 18C shows raw and average ultrasound-evoked multi-unit activity(MUA) recorded from M1 cortex.

FIG. 18D shows an approach to stimulating descending corticospinaltracts with transcranial ultrasound.

FIG. 18E shows electromyogram (EMG) traces for a spontaneous andultrasound-evoked event.

FIG. 19A shows plot of the electromyogram response latency of lefttriceps brachii in response to right M1 activation as function ofrepetitive trial number.

FIG. 19B shows electromyogram failure probability histograms for fourprogressively decreasing ITIs.

FIG. 19C shows raw electromyogram traces illustrating application oftetrodotoxin (TTX) to the motor cortex blocks ultrasound-evokeddescending corticospinal circuit activity.

FIG. 19D shows temperature recordings from M1 in response totransmission of ultrasound waveforms.

FIG. 19E shows normalized ultrasound-evoked electromyogram amplitudehistograms plotted for four ultrasound frequencies.

FIG. 19F shows normalized ultrasound-evoked electromyogram amplitudesplotted as function of ultrasound intensities.

FIG. 20A shows histograms for mean synaptic density, mean axonal boutonsynaptic vesicle density, mean postsynaptic density length, and meannumber of docked vesicles occupying active zones.

FIG. 20B shows histograms for the mean density of cleaved-caspase 3positive glial cells and neurons in the motor cortex of control andultrasound-stimulated hemispheres.

FIG. 20C shows histograms for mean rotorod running times and mean wirehang times at 24 h pre-treatment and 24 h and 7 d post-treatment.

FIG. 21 shows the strategy for constructing typical low-intensityultrasound waveforms.

FIG. 22 shows attenuation of ultrasound stimulus waveforms bytranscranial transmission.

FIG. 23A shows a spike raster plot that illustrates an increase incortical spikes as a function of time in response to ultrasoundstimulation.

FIG. 23B shows extracellular neuronal activity traces recorded inresponse to ultrasound stimulation.

FIG. 23C shows a post-stimulus time histogram illustrating the averagemulti-unit activity (MUA) spike count before and after ultrasoundstimulation.

FIG. 24A shows a histogram illustrating the mean density of c-fospositive cells from ultrasound-stimulated hemispheres as a function oftargeted region.

FIG. 24B shows a histogram illustrating the mean area of clusterscontaining c-fos positive cells.

FIG. 25 shows normalized pressure profiles following ultrasoundstimulation.

FIG. 26A shows an illustration depicting some of the proposed fluidmechanical actions by which ultrasound can modulate neuronal activity,

FIG. 26B shows an illustration of a composite model of brain tissue,where different cellular interfaces establish boundary sites havingdifferent properties due to acoustic impedance mismatches.

FIG. 27A shows an illustration of 10 cycles of a sine wave-generatedultrasound pulse at 0.50 MHz.

FIG. 27B shows an illustration of 10 cycles of a sine wave-generatedultrasound pulse at 0.25 MHz.

FIG. 27C shows an illustration of 10 cycles of a square wave-generatedultrasound pulse at 0.25 MHz.

FIG. 28A shows an illustration of the repetition of 10 cycles of sinewave-generated ultrasound pulses at 0.50 MHz.

FIG. 28B shows an illustration of the repetition of 10 cycles of sinewave-generated ultrasound pulses at 0.25 MHz.

FIG. 28C shows an illustration of the repetition of 10 cycles of squarewave-generated ultrasound pulses at 0.25 MHz.

FIG. 28D shows an illustration of the repetition of alternating 10cycles of sine wave-generated ultrasound pulses at 0.50 and 0.25 MHz.

FIG. 28E shows an illustration of the repetition of alternating 10cycles of square wave-generated ultrasound pulses at 0.25 MHz and sinewave-generated ultrasound pulses at 0.50 MHz.

FIG. 28F shows an illustration of the repetition of alternating 10cycles of sine wave-generated ultrasound pulses at 0.25 MHz and squarewave-generated ultrasound pulses at 0.20 MHz.

DETAILED DESCRIPTION

The present invention comprises methods and devices for modulating theactivity of cells. The methods and devices comprise use of ultrasoundwaves directed to cells which may be found in cell cultures or in vivoin living bodies. Methods of the present invention comprise use of theapplication of ultrasound waves, such as low intensity, low frequencyultrasound, or low intensity ultrasound, to living cells to affect thecells and modulate the cells' activities. Devices of the presentinvention comprise one or more components for generating ultrasoundwaves, including but not limited to ultrasonic emitters, transducers orpiezoelectric transducers, composite transducers, CMUTs (capacitivemicromachined ultrasound transducers), and which may be provided assingle or multiple transducers or in array configurations. Theultrasound waves may be of any shape, and may be focused or unfocused,depending on the application desired. The ultrasound may be at anintensity in a range of about 0.0001 to about 900 mW/cm² and anultrasound frequency in a range of about 0.02 to about 1.0 MHz at thesite of the cells or tissue to be modulated.

As disclosed herein, aspects of the invention are described in thecontext of providing ultrasound to mammalian brain tissue in vitro andin vivo. However, the invention is not limited to this context. Aspectsof the invention comprise providing ultrasound to cells, where everlocated in a living body, such as human, animal, insect, avian bodies,or to cells found in cell culture, or microbial or one celled organisms.For example, the activity of neural tissue may be modulated in vivo inthe brain or elsewhere in the body of a living organism, or in an invitro sample mounted in a vessel of any kind for any purposes, includingelucidating the functioning of normal or disordered neural tissue, ordiagnosing or treating a neural disorder in a living organism. As usedherein a neural disorder includes any functional or physiologicalabnormality or injury or psychiatric disorder, such as stress anddepression. As used herein neural tissue includes tissue with neuronswithin it, or neural precursor cells, such as neural stem cells,neurons, axons, neural cell bodies, ganglia, dendrites, synapticregions, neuronal tissue, or other cells positioned in a living organismamong neurons, such as glial cells, oligodendrites, or astrocytes.Treatment of neural tissue is exemplary and is not intended to limit theinvention.

Ultrasound has been shown to influence neuronal activity by suppressingthe amplitudes and/or conduction velocity of evoked action potentials.Detailed investigations are lacking however and the underlyingmechanisms of these effects remain unknown. Moreover, nearly all ofthese previous studies examining the effects of ultrasound on neuronalactivity have implemented long irradiation times (minutes) withhigh-frequency (>1 MHz) ultrasound delivered at moderate intensitylevels (>500 mW/cm²). The use of moderate and high intensity,high-frequency ultrasound and long exposure times to control neuronalactivity minimizes ultrasound's practicality for modulating neuronalactivity in living organisms. The present invention comprises methodsfor low-intensity (<500 mW/cm²), low-frequency ultrasound (<0.9 MHz) andeffects on cellular modulation, such as methods for influencing neuronalactivity. For example, low intensity may comprise about 450 mW/cm², 400mW/cm², 350 mW/cm², 300 mW/cm², 250 mW/cm², 200 mW/cm², 150 mW/cm², 100mW/cm², 50 mW/cm², 25 mW/cm², 10 mW/cm², and levels of ultrasoundintensity within these stated amounts, including from about 450 mW/cm²to about 1 mW/cm². Low frequency ultrasound may comprise ranges fromabout 0.88 MHz to about 0.01 MHz, from about 0.80 MHz to about 0.01 MHz,0.80 MHz to about 0.1 MHz, from about 0.70 MHz to about 0.1 MHz, fromabout 0.60 MHz to about 0.1 MHz, from about 0.50 MHz to about 0.1 MHz,from about 0.40 MHz to about 0.1 MHz, from about 0.30 MHz to about 0.1MHz, from about 0.20 MHz to about 0.1 MHz, from about 0.10 MHz to about0.1 MHz, and levels of ultrasound frequency within these stated amounts.

As used herein, the cited intensities and frequencies are the intensityand frequency levels at the effective tissue site, not the actual outputnumber of the transducer. For example, the pressure waveform experiencedat the site of the target tissue is below about 0.9 Mhz or 900 mW/cm².The output of a transducer may have to be much larger than the resultingeffective amount at the target tissue site. For example, a transducermay output 90 W for transmission to an intact skull for the effectiveamount at the brain to be below about 0.9 Mhz or 900 mW/cm², as theskull absorbs a significant portion of ultrasound waves. Thus, thefrequencies and intensities stated and claimed herein are thefrequencies and intensities experienced at the target tissue site, notthe output of the ultrasound transducers.

As used herein, providing ultrasound treatment or ultrasound to a targetsite to modulate cellular activity comprises providing an ultrasoundstimulus waveform to a subject. The ultrasound stimulus waveform mayalso alternatively be referred to herein as a waveform, and the twoterms are used interchangeably as can be understood by those skilled inthe art. A stimulus waveform may be provided to a subject, human, animalor other subjects, once or multiple times in a single treatment, or in acontinuous treatment regimen that continues for a day, days, weeks,months, years, or for the life of the subject. Determining the length oftreatment needed is within the skills of medical and/or researchprofessionals. It is contemplated by the present invention that astimulus waveform may be pulsed or continuous, have one or multiplefrequencies, and other characteristics as described herein. For example,in particular treatments, a pulsed ultrasound stimulus waveform may betransmitted for about 10 microseconds, for about 25 microseconds, forabout 50 microseconds, for about 100 microseconds, for about 250microseconds, for about 500 microseconds, for about 1000 microseconds,for about 2000 microseconds, for about 3000 microseconds, for about 4000microseconds, for about 5000 microseconds, for about 1 second, for about2 seconds, for about 3 seconds, for about 4 seconds, for about 5seconds, for about 6 seconds, for about 7 seconds, for about 8 seconds,for about 9 seconds, for about 10 seconds, and then this treatment maybe repeated for the same or a different length of time, one or moretimes. For example, a stimulus waveform may be provided every 11 secondsfor a duration of about 250 microseconds for years, or for the life ofthe subject.

FIG. 1 is a block diagram that illustrates an example system 100 formodulating cellular activity, according to an embodiment wherein thetarget site is neural tissue. To illustrate the operation of system 100,a body 190 with an external surface 192 that encompasses neural tissue194 is depicted. However, the system 100 does not include the body 190or its external surface 192 or neural tissue 194. In some embodiments,the body 190 is a living organism, such as a human or animal or othersubject, or a portion thereof, such as a head and skull. In someembodiments, the body is a vessel that contains an in vitro sample, suchas a glass cylinder filled with water or artificial cerebrospinal fluidand a suspended slice extracted from the brain of an organism.

The system 100 includes components for generating ultrasound waves suchas ultrasound transducers 110, including transducer 110 a and transducer110 b, and controller 150. In some aspects, the transducer may be anemitting transducer, a receiving and transmitting transducer, or areceiving transducer. The ultrasound transducers 110 are connected tocontroller 150 for receiving waveform and power, and the transducers aredriven by the controller. The transducers are acoustically coupled tothe external surface 192 of body 190 in order to introduce acousticenergy into the body 190. The transducers 110 use the received waveformand power to emit ultrasound frequency acoustic beams 120, such as beam120 a from transducer 110 a and beam 120 b from transducer 110 b. Thecontroller 150 includes a process 154 for waveform formation, whichdetermines the waveform to be emitted by transducers 110 a into body190. In some embodiments, the transducers are battery powered andreceive only waveform information from controller 150.

Although a particular number of transducers and controllers are depictedin FIG. 1 for purposes of illustration, in other embodiments, more orfewer or the same number of transducers is included, and controller 150is replaced by one or more devices that each perform a different orredundant function of controller 150, including the waveform formationprocess 154. Although FIG. 1 depicts separate wired connections betweentransducers 110 and controller 150 to send power and waveforms totransducers 110, in other embodiments one or more connections may bewireless, or carry power or waveforms for multiple transducers 110.

In the illustrated embodiment, the two transducers 110 each transmit anacoustic beam into body 190, which intersect in beam intersection region122. In some embodiments, the waveform transmitted in a beam iseffective in modulating neural activity everywhere the beam intersectsthe neural tissue. In some embodiments, the waveform transmitted in abeam is only effective (or more effective) in an intersection region 122with another beam. In some embodiments, the transmitted waveforms areeffective in only a portion of the intersection region 122, dependentupon interference patterns of constructive and destructive interferenceamong the waveforms in the intersecting beams.

The intensity of the acoustic beam is given by the amount of energy thatimpinges on a plane perpendicular to the beam per unit time divided bythe area of the beam on the plane, and is given in energy per unit timeper unit area, i.e., the power density per unit area, e.g., Watts persquare centimeter (W/cm²). This is the spatial-peak temporal-averageintensity (Ispta); and is used routinely for intensity hereinafter. Inillustrated embodiments, the Ispta is less than 500 mW/cm². Anotherdefinition of intensity widely used in the art is spatial-peakpulse-average intensity (Ipa); for the multiple cycle pulses used in theillustrated embodiment the Ipa is typically less than 10 W/cm².

Any means known in the art may be used to transmit an acoustic beam 120into a body 190. For example, Archimedes SI transducers (ArrayTherapeutic, Scottsdale, Ariz., USA) may be used, which are a type ofpiezo-electric transducers (PZT). An Archimedes SI has two peak responsefrequencies at which the transmitted acoustic pressure is 71% (−3 dB) ofits maximum value. For example, Archimedes transducers had one peak at0.44 MHz and another at 0.67 MHz. Other ultrasound transducers may beused, including but not limited to, Olympus NDT/Panametrics 0.5 MHzcenter frequency transducers, as well as Ultran 0.5 and 0.35 MHz centerfrequency transducers.

In some embodiments, capacitive micro-machined ultrasonic transducer(CMUT) technology may be applied. For example, CMUTs may be arranged inflexible array designs that comfortably permit adaptive beam forming andfocusing. And in other embodiments the CMUTs may be mounted inside abody cavity to transmit ultrasound to cells, tissues, or organs.Furthermore, CMUTs may be mounted to the skull to transmit ultrasound tovarious brain regions.

Any devices known in the art may be used in controller 150. In anillustrated embodiment, waveforms were generated using an Agilent 33220Afunction generator (Agilent Technologies, Inc., Santa Clara, Calif.,USA) and amplified using an ENI 240L RF amplifier. Pulses in somewaveforms were triggered using a second Agilent 33220A functiongenerator. Data controlling the above devices may be generated bywaveform formation process 154 using a general purpose computer withsoftware instructions, as described in more detail in a later section.

Although system 100 is depicted with two transducers and correspondingbeams, more or fewer transducers or beams or both may be included in asystem.

Systems and devices for providing ultrasound for the present inventionmay comprise materials that bend light or sound and can focus the waves.Such materials have been used to make super-lenses. Such materials,super-lenses and other similar components may be used to focus theultrasound waves in the methods and devices of the present invention.For example, transducers, of any type, in conjunction with a focusingelement such as a super-lens or metamaterial are used for focusing theultrasound waves used to modulate cellular activity. Such materials canrefract light backward, or have a negative index of refraction and havebeen referred to as a “metamaterial.” An example of a metamaterial is asound-focusing device comprising an aluminum array of narrow-neckedresonant cavities with dimensions that are tuned to interact withultrasound waves. The cavities may be filled with water. A focusingelement, such as a metamaterial, may be used in conjunction with one ormore transducers, and/or with phased arrays of transducers.

FIG. 2 is a graph that illustrates an example ultrasound waveform 200for modulating neural activity, according to an embodiment. Thehorizontal axis 202 indicates time, and the vertical axis 204 indicatespressure, both in arbitrary units. The modulating waveform 200 containsone or more pulses, such as pulse 220 a and pulse 220 b and pulse 220 c.Each pulse includes one or more cycles at an ultrasound frequency. Forexample, pulse 220 a includes five cycles of an ultrasound frequencywith a period (τ) 210 in seconds equal to the reciprocal of thefrequency (f) in Hertz (i.e., τ=1/f). The number of cycles in a pulse isdesignated cycles per pulse (c/p). The pulse length 222 is designated PLand is given in seconds by the product of the period r and number ofcycles per pulse c/p, i.e., PL=τ*c/p.

Pulses are separated by quiescent periods that are related to the timebetween pulse starts, shown in FIG. 2 as pulse repeat time 230. Thereciprocal of the pulse repeat time 230 in seconds is the pulse repeatrate in Hertz, designated herein the pulse repeat frequency PRF, todistinguish it from the ultrasound frequency f. In some embodiments, thepulse repeat frequency PRF is a constant for a waveform 200. In someembodiments, the pulse repeat frequency PRF increases from a minimum(PRF min) to a maximum (PRF max) over a time interval called a ramptime. For example, in some embodiments, PRF increases from PRF min=0 toPRF max=3000 Hz over ramp time=5 seconds. In other embodiments the PRFmay range from 0.001 to 10 KHz. The waveform continues for a waveformduration 234 that ends with the last pulse n the wave form. The numberof pulses in the waveform is designated Np.

The pressure amplitude of the ultrasound wave is proportional to avoltage range used to drive a piezoelectric transducers (PZT). Forexample, in the illustrated embodiments, the voltage range is selectedbetween 100 milliVolts (mV, 1 mV=10⁻³ Volts) and 500 mV, whichcorrespond to intensity levels less than 500 mW/cm². Although pulses areshown in FIG. 1 as sine waves having a single ultrasound frequency, invarious other embodiments, other oscillating shapes may be used, such assquare waves, or a pulse includes multiple ultrasound frequenciescomposed of beat frequencies, harmonics, or a combination of frequenciesgenerated by constructive or deconstructive interference techniques, orsome or all of the aforementioned.

FIG. 3 is a flow diagram that illustrates, at a high level, a method 300for modulating neural activity according to an embodiment. Although aparticular number of steps are shown in a particular order for purposesof illustration, in other embodiments, one or more steps may beperformed in a different order or overlapping in time, in series or inparallel, or one or more steps may be omitted or added, or changed insome combination of ways.

In step 310, one or more ultrasound transducers are acoustically coupledto an outside surface of a body which may include or encompass neuraltissue, or other tissues. In some embodiments, one or more transducersare phase transducer arrays. In some embodiments the body is a vesselwith artificially produced brain fluid in which is suspended a slice ofneural tissue. In several of these embodiments, the coupling is directcontact of an ultrasound piezoelectric hydrophone with the fluid in thevessel, or a mechanical coupling of a piezoelectric material to a wallof the vessel. In therapeutic embodiments, the body is a patient and thetransducers are acoustically coupled to the skin of the patient, such ason the head or back. In some embodiments, acoustic coupling is affectedby a gel or other substance, well known in the art, which prevents lossof acoustic energy to the air surrounding the patient. In someembodiments, step 310 includes shaving hair from a portion of apatient's head. In some embodiments, air-coupled ultrasound transducerstransmit ultrasound pulses through the air in a manner to target theneural tissue by penetrating the skin, bone, muscle, and underlyingfascia. In some embodiments, one or more ultrasound transducers may bebolted directly to a structure such as the skull underneath the skin. Insome embodiments, ultrasound transducers may be mounted inside thecavity of a patient, such as in the peritoneal or thoracic cavity.

In step 320, the one or more transducers (or phase transducer arrays)coupled to the body are driven at ultrasound frequencies and lowintensity in multiple short pulses that are effective in modulatingneural activity in at least a portion of the neural tissue. For example,the beam intersects only a portion of the neural tissue, or multiplebeams intersect in a portion of the neural tissue and only tissue inregions of constructive and or deconstructive interference is modulated.It is noted that the scale of constructive interference patters ismillimeters based on the wavelength of ultrasound in neural tissue. Forexample, the speed of sound in water approximates the speed of sound insoft body tissue and is about 1500 meters per second. Thus at ultrasoundfrequencies from 0.1 to 1 MHz, the wavelength of ultrasound is betweenabout 1.5 mm and about 15 mm. Constructive and or deconstructiveinterference patterns are on the same order as these wavelengths.

Ultrasound frequencies may be selected to penetrate to the neural tissueor target tissue. For samples suspended in vessels and transducersattached to the outside all of the vessel, the ultrasound frequency isselected in a range that effectively passes through the vessel (e.g.,glass) wall with little attenuation. For transducers that directlycontact the brain fluid, penetrating a different material is not asignificant issue. For transducers placed on a patient's head, theultrasound frequency should pass through the skull with littleabsorption to prevent heating the skull, which can cause discomfort orinjury to the patient. It has been found that ultrasound frequenciesbetween about 0.2 MHz and about 0.9 MHz pass through the skull withlittle deleterious heating even at high intensity if proper coolingprecautions are implemented. The ultrasound intensity is chosen to havea modulating effect on neural tissue without damage to the tissue. Ithas been found that intensities below about 500 mW/cm² are effective inmodulating neural activity without detected damage to neurons and othercells in brain tissue.

In step 330, an effect on neural activity is determined. In variousembodiments, the effect is stimulation or suppression of neuralactivity. For example, in some embodiments, an increase or decrease influorescence that indicates neural activity is detected. In someembodiments, a membrane voltage change is detected. In various otherembodiments, other phenomena that reflect neural activity are monitored,such as Positron Emission Tomography (PET) scans and brain metabolitessignatures in nuclear magnetic resonance imaging (MRI) scans or othermeasures of neural activity such as electroencephalogram (EEG) ormagnetoencephalography (MEG). In therapeutic embodiments, a change in aprogression or symptom of a disease or disorder is determined. In someembodiments, for example in embodiments in which therapeuticeffectiveness is well established, step 330 may be omitted.

FIG. 4A is a block diagram that illustrates an example experimentalsetup 400 to demonstrate effects on neural activity from an ultrasoundwaveform, according to an embodiment. The setup 400 includes a vesselthat contains artificial cerebrospinal fluid (aCSF) 490 and neuraltissue slice 494 loaded with a fluorescent marker that is used to detectneural activity. An ultrasound transducer 410 is coupled to the aCSF andintroduces one or more low power ultrasound pulses 422 in an acousticbeam 420 directed onto the neural tissue slice 494. The slice is mounteda distance from the transducer that is indicated in FIG. 4 by the aCSFcolumn 492. The setup includes a laser-scanning confocal imagingmicroscope 480 that views the neural tissue before, during and afterultrasound exposure to detect neural activity changes.

In some embodiments, a whole brain is mounted in the aCSF 490 in placeof the slice 494. To determine the acoustic intensity impinging at thelocation of slice 494, in a calibration step performed in someembodiments of step 320, a hydrophone replaces the slice 494 while thetransducer 410 is driven. The height of aCSF column 492 is adjusted invarious embodiments. For example, in various embodiments, the height ofcolumn 492 was varied from 4.5 to 45 mm.

FIG. 4B is a graph 430 that illustrates an example acoustic signal 436received at a location of neural tissue, according to an embodiment, asmeasured by a hydrophone. The horizontal axis 432 indicates time inmicroseconds (μs, 1 μs=10⁻⁶ seconds). The vertical axis 434 indicatespressure in MegaPascals (MPa, 1 MPa=10⁶ Pascals=10⁶ Newtons per squaremeter and is about ten time atmospheric pressure). The signal 436 wasmeasured at height about 2 mm above transducer 410 which was driven atultrasound frequency 0.44 MHz in a single pulse including 10 cycles andvoltage range of 500 mV p-p pulses and further amplified using a 50 dBgain RF amplifier.

FIG. 4C is a graph 440 that illustrates an example spectrum 446 of theacoustic signal depicted in FIG. 4B, according to an embodiment. Thehorizontal axis 442 indicates frequency in MegaHertz. The vertical axis444 indicates amplitude in arbitrary units. The spectrum 446 wascomputed from the pressure signal 436 using a digital Fast FourierTransform (FFT), and contains one salient peak 447 at 0.44 MHz, theultrasound driving frequency for transducer 410.

In several illustrated embodiments, an ultrasound waveform designatedUSW-1 consists of 250 ultrasound pulses, each pulse consisting of 10square wave cycles at 0.44 MHz, for a pulse length of 22.7 microseconds.The pulse repeat frequency (PRF) ramps up over five seconds from PRFmin=0 to PRF max=100 Hz (thus averaging 50 pulses a second over thefirst 5 seconds). The total waveform duration is therefore 5 seconds.The peak to peak square wave amplitude driving the transducer of theillustrated embodiment was 500 mV. The corresponding pulse averageultrasound intensity is 23 mW/cm².

According to an embodiment, histology can be used to observe modulatedneural activity. For example, at the end of the waveform, 5 secondsafter the start of modulation by USW-1, fluorescent emissions in CA1 SPand CA1 SR regions are greater than fluorescent emissions in the sameregions at 2 seconds before modulation by ultrasound waveform. Such ahistological comparison indicates significant modulation of neuralactivity by this ultrasound waveform at this low intensity.

FIG. 5 is a graph 530 that illustrates example temporal response ofneural activity after modulation, according to an embodiment. Thehorizontal axis 532 is time in seconds; and the horizontal scale isgiven by segment 531 that corresponds to 10 seconds. The vertical axis534 indicates ΔF in percent (%); and the vertical scale is given bysegment 535 that corresponds to 10%. The start of USW-1 is indicated bytick 533. Individual responses after each ultrasound waveform are givenby traces 540. A thick light curve 540 indicates the average temporalresponse. As can be seen in graph 530, the neural activity indicated byspH increases strongly during the five second duration of the ultrasoundwaveform up to almost 20%, and stays elevated, though at an everdiminishing level, for more than 10 seconds after the waveform ends.This indicates significant and persistent modulation of neural activityby this ultrasound waveform at this low intensity.

FIG. 11 is a bar graph that illustrates example enhanced effects onneural activity by modulation with particular ultrasound waveforms,according to several embodiments. The horizontal axis indicatesdifferent low intensity waveforms, at intensities less than 500 mW/cm².The vertical axis indicates the mean peak ΔF obtained for many neuronsin response to the waveform, normalized by the mean peak ΔF obtainedfrom many neurons in response to USW-1.

Bar 1210 indicates the normalized ΔF for USW-1 and is equal to 1 bydefinition. Recall that USW-1 is characterized by f=0.44 MHz, c/p=10,Np=250, PRF=ramp (for a waveform duration of 5 seconds). Bar 1220indicates the normalized ΔF for a waveform (called USW-1220 hereinafter)characterized by f=0.44 MHz, c/p=80, Np=10, PRF=10 Hz (for a waveformduration of 1 second). Bar 1230 indicates the normalized ΔF for awaveform (called USW-1230 hereinafter) characterized by f=0.44 MHz,c/p=80, Np=3, PRF=10 Hz (for a waveform duration of only 0.3 seconds).Bar 1240 indicates the normalized ΔF for a waveform (called USW-1240hereinafter) characterized by f=0.44 MHz, c/p=80, Np=30, PRF=10 Hz (fora waveform duration of 3 seconds). Bar 1250 indicates the normalized ΔFfor a composite waveform (called USW-1250 hereinafter) characterized bymultiple ultrasound frequencies, cycles per pulse and pulse repeatfrequencies, as described in more detail below.

USW-1 produces effects comparable to therapeutic effects produced byelectrical stimulation from surgically implanted electrodes. As can beseen in graph 1200, several waveforms produce effects, about three timesthe effect of USW-1 and more, without the intrusion and danger ofimplanted electrodes.

The effects of USW-1220 and USW-1230 show that fewer pulses at the samefrequency are effective. The effect of USW-1240 at the same ultrasoundfrequency but longer duration is slightly diminished, and the effect ofUSW-1 at the same frequency but even longer duration is substantiallydiminished, suggesting a saturation effect on particular cell membranecomponents affected by this ultrasound frequency.

To avoid such saturation, USW-1250 changes several aspects of thepulses. Specifically USW-1250 is characterized by f=0.22 MHz, c/p=10,Np=25, PRF=ramp from 0 to 50 Hz over 1 second; followed by f=0.44 MHz,c/p=10, Np=150, PRF=ramp from 0 to 100 Hz over 3 seconds; followed byf=0.67 MHz, c/p=7, Np=150, PRF=ramp from 0 to 150 Hz over 2 seconds (fora waveform duration of 6 seconds). USW-1250 produces nearly four timesthe effect of USW-1 and none of the saturation suggested by USW-1240 andUSW-1.

Other waveforms with multiple pulse characteristics are effective inmodulating neural activity and cellular activity. Another effectivewaveform is characterized by f=0.44 MHz, c/p=10 Np=10, PRF=1 Hz (for aduration of ten seconds). This waveform produced depression of neuralactivity in experiments.

The present invention comprises methods and devices for modulatingcellular activity in a subject. Methods comprise providing ultrasound toa subject, for example, by the use of one or more low intensity, lowfrequency ultrasound and/or low intensity ultrasound transducers. Forexample, the ultrasound (US) transducer can be acoustically coupled toan external surface of a subject, or alternatively, the US transducercan be in an acoustically effective range of the target tissue, and theultrasound transducer can then be driven to form stimulus waveforms inthe tissue, cell, or organ with an intensity below about 900 milliWattsper square centimeter (mW/cm²). The ultrasound waveforms may compriseone or multiple frequency components.

In an embodiment, driving the ultrasound transducer further comprisesdriving the ultrasound transducer to form pressure fluctuation waveformor a stimulus waveform including a plurality of pulses, each pulse ofduration less than about 10000 microseconds (μs). Pulse duration may bevariable depending on a particular method or device, and may have aduration of about 10 seconds or less, such as about 100 to 10000microseconds. Driving the ultrasound transducer can further comprisedriving the ultrasound transducers to form a pressure fluctuationwaveform or a stimulus waveform with a plurality of pulses within awaveform duration that is less than about ten second (s). This comprisesonly one stimulus waveform and this waveform maybe repeated a nearlyinfinite number of times. As used herein, pressure fluctuation waveformand stimulus waveform are used interchangeably.

Driving the ultrasound transducer may further comprise driving theultrasound transducers to form a stimulus waveform at a frequency aboveabout 0.20 MHz. The waveform may be one or more of known waveformsarbitrary or not, including but not limited to, sine, square, sawtoothand triangle. The ultrasound waves may be focused to provide action at aparticular site in or on the subject, or the waves may be unfocused andprovide action at multiple sites. The waves may be continuous or pulsed,depending on the desired application. The frequency or intensity may beuniform throughout a treatment period, or may alternate or sweep fromone number to another, and back to the original number. Those skilled inthe art are able to determine such parameters for the desiredapplication. Examples are disclosed herein.

The low-intensity, low-frequency ultrasound described herein can be usedto stimulate cellular molecular pathways in cells, and for example, tocause them to (i) secrete signaling molecules (i.e., insulin from betacells, BDNF, GDNF from neurons and glia, CCK from intestinal cells etc),(ii) increase cell proliferation, (iii) induce cell differentiation,(iv) modulate transcription, (v) modulate translation, or (vi) acombination thereof. These actions can involve a calcium-dependentprocess. For example, calcium can come from intracellular stores such asIP3, RyR and TRP, or from other membrane ion channels, such as voltagesensitive, mechanosensitive, and TRP channels.

The use of low-intensity, low-frequency ultrasound can activate calciumsignaling pathways for the use of various therapies in varioustissue/cell types (such beta cells in pancreas for treatment ofdiabetes, cardiac cells for treatment of heart disorders and others, asdescribed herein). The action of the ultrasound described herein canfunction in both neural and non-neural cells to activate signalingpathways, and thereby have a therapeutic value. For treating traumaticbrain injury, for example, methods comprise using ultrasound tostimulate the release of neuroprotective agents without causing cells tofire action potentials, although ultrasound may be used induce actionpotentials, if desired. For a treatment of diabetes, methods compriseproviding ultrasound for the stimulation of insulin secretion (throughdirect actions on the beta cells or through neurostimulation of vagalefferents), as well as causing beta cells to proliferate by stimulatingthe pancreas directly with ultrasound.

The methods and devices of the present invention may modify the fluiddynamics of organs and fluids within organs, such as brain fluids and/orviscoelastic neuronal membranes, in a manner that increases or decreasesneuronal activity. The ultrasound treatments of the present inventionmay modify channels to regulate ions, as well as fluid mechanics in amanner to then directly modify the activity of cells residing in theextracellular fluids or ultrasound waves may modify the fluid dynamicsand membrane permeability of cells directly. Providing ultrasound wavesto a target tissue may modify the fluid dynamics of that target tissuesuch that is affected. US may act on extracellular and intracellularfluids, as well as on cell membranes themselves, resulting in at leastmodifying the activity of cells.

Modulation of cellular activity by ultrasound may comprise a change inthe secretion of signaling molecules, the proliferation of cells, thedifferentiation of cells, the modulation of protein transcription, themodulation of protein translation, modulation of proteinphosphorylation, modulation of protein structure such as by changing theprotein structure itself or through dimerization or other formation ofprotein multimers, activating caspases or other proteins, or acombination thereof. Such changes in cellular activity or activities maybe detected in the cells themselves, in the results of such cellularchanges on structures such as changes in nerve activity, or otherphysical changes such as altered insulin use or release by cells,restored brain activity or cessation of brain activity, or otherphysical parameters that may be measured for the treated subject.Methods for detecting cellular activity change are known to thoseskilled in the art. Such tests include tests from the molecularbiological level to gross anatomical determinations, for example, DNAtranscription rates, protein phosphorylation changes, and physiologicaldeterminations such as blood tests, hormone release and usage tests,nerve function tests, and subject reports of health, pain, cessation orincrease of desires or drives.

An aspect of the present invention comprises modulating cellularactivity, such as neural cells or other cells, by providing pulsed orcontinuous ultrasound waveforms having arbitrary or particular forms. Amethod of the present invention comprises modulating cellular activityin a noninvasive manner by transmitting specific sets of pulsedultrasound (US) waveforms, for example, to intact neuronal circuits.Though not wishing to be bound by any particular theory, it is currentlybelieved that spatiotemporal pattern(s) of the US energy itself, as wellas its actions on a neuronal circuit may be related to modulatingneuronal activity. The pulse sequence(s) delivered to the tissue may berelated to successful stimulation/inhibition of neuronal activity. Thepresent invention comprises methods and devices for modulation of cells,including neuronal cells, in brain slices, whole ex vivo brains, andintact brain circuits. Such methods and devices may comprise differentultrasound transducer types, and may be acquired from differentmanufacturers, for example, Array Therapeutic, LLC, OlympusNDT/Panametrics, and Ultran. The effects of the treatments and methodsdescribed herein are not dependent on the device, equipment, or brainpreparation, but on the ultrasound delivery.

An aspect of the present invention comprises in vivo use of US pulseshaving lower pulse intensity integrals when compared to those used invitro. To achieve a temporal average intensity similar to those used invitro, US pulses were delivered to intact brain with a higher pulserepetition frequency than those used in vitro. Intensities ranging fromabout 0.0001 to 900 mW/cm² with frequencies (single ormultiple-component; see FIGS. 27 and 28 ) ranging from about 0.1 to 0.9MHz are effective for modulating neuronal activity. An aspect of theinvention comprises using temporal average intensity (I_(TA)) for USpulses of greater than 20 mW/cm², and about 50 mW/cm² at the cellularsite, are useful for stimulating neuronal activity. An aspect of theinvention comprises using intensity values less than about 100 W/cm² andfrequencies less than about 0.9 MHz occurring at the neural circuitbeing targeted for modulating neuronal activity in the intact brain ofan organism.

The present invention comprises use of low-intensity, low-frequencypulsed ultrasound for affecting modulation of cells. US may be providedalone or with other agents. Such other agents may be provided to thesubject before, during or after US exposure. Such agents include, butare not limited to, exogenous genes, chemicals, proteins,pharmaceuticals, gases, antibiotics, substrate molecules, ionicmolecules, or other active agents.

Pulsed US waveforms useful for modulating cellular activity such asneuronal activity may be created using different methods. For example,neuronal circuits can be excited in a clear and robust manner usingpulsed ultrasound having <0.9 and >0.2 MHz. For example, it has beenfound that a continuous wave of US at a frequencies 0.2 to 0.9 MHz donot produce neuronal stimulation, but rather inhibition of activity suchthat no responses are obtained. An aspect of the invention comprisesusing US in a specific set of pulse sequences for the stimulation ofneuronal activity within that frequency range. The US may be deliveredin focused ultrasound pulses, or may be delivered as collimated orunfocused planar waves. Such ultrasound waves may be delivered usingsingle ultrasound components, such as a transducer, or from 1 to 299transducers, and some component devices may contain up to 1000transducers for focusing and resolution control.

An aspect of the present invention comprises stimulating brain activity,or other cellular modulation, by using US waveforms constructed ofdistinct US pulses (from 1 to 50,000 cycles) having a particularfrequency and then alternating the length and frequency of US pulses sothat the entire stimulus waveform is composed of US pulses havingvarying fundamental frequencies and durations. Though not wishing to bebound by any particular theory, it is currently believed that byalternating the frequencies of US within a stimulus waveform it keepsthe neuronal membrane and its environment from adapting to acousticpressure changes, which then permits more effective and robustmodulation compared to US waveforms composed of a single fundamentalfrequency.

For example, methods of the present invention for intact brain circuits,in vivo, used ultrasound PZT transducers (manufactured by Ultran and/orOlympus NDT/Panametrics), with a mean optimal resonant center frequencyof 0.5 MHz. To create US stimulus waveforms which had multiple frequencycomponents, transducers were driven at a fundamental frequency off ofthe center frequency using square-wave voltage pulses, which produced USstimulus waveforms having various beat frequencies and harmonicfrequencies.

The present invention contemplates US pulses as shown in FIGS. 27 and 28, which may be driven using 10 cycle voltage pulses, but that the numberof voltage cycles driving transducers can range from 1 to 50,000 orhigher. The number of US pulses per US waveform may similarly range from1 to 10000000 repeated at pulse repetition frequencies from 0.0001 to100 MHz for a total stimulus waveforms duration from about 0.000001 secto a continuous application of the stimulus wave.

FIG. 27A illustrates a 10 cycle US pulse generated using a 0.5 MHz 10cycle sine wave voltage pulse delivered to the transducer. Thetransducer has a center frequency of 0.5 MHz. The FFT power spectrum(right) of this waveform, shows a single peak representing thefundamental frequency of 0.5 MHz. FIG. 27B similarly illustrates a 10cycle US pulse generated using a 0.25 MHz 10 cycle sine wave pulsedelivered to the same transducer. The FFT power spectrum at right a peakcorresponding to the fundamental frequency (0.25 MHz), as well as theharmonic (0.75 MHz) is observed. Driving the transducer off its centerfrequency introduces off-resonant energy. US stimulus waveforms composedof mixed frequency components appeared may be effective for manyapplications of the methods and devices of the present invention. Forexample, transducers using square waves at fundamental frequencies offof their center frequency are shown in FIG. 27C. FIG. 27C illustrates a10 cycle US pulse generated using a 0.25 MHz 10 cycle square wave pulsedelivered to the same transducer as above. The FFT power spectrum, atright, has a peak corresponding to the fundamental frequency (0.25 MHz),as well as a beat frequency (0.5 MHz) and the harmonic (0.75 MHz) can beobserved. Thus the US pulse itself introduces multiple frequencycomponents.

The present invention contemplates methods comprising ultrasound pulsescomposed of a fundamental frequency, and alternating with US pulseshaving different fundamental frequencies. The present inventioncontemplates methods comprising US pulses composed of multiplefrequencies in a stimulus waveform by generating US pulses havingdifferent frequency components rather than only the fundamentalfrequency. For example, the same US pulse may be repeated across time atsome pulse repetition frequency, rather than delivering different pulsescomposed of different fundamental frequencies. Driving the transducerswith a square wave having a fundamental frequency matched to the centerfrequency does not produce the type of effect as illustrated in FIG.27C. An aspect of the invention comprises methods wherein square wavesare used, but the transducers are driven very close to their centerfrequency, for example where the transducer has two peak frequencies0.44 and 0.67 MHz. Pulse sequences as illustrated in FIG. 27D result,but at different frequencies more closely matching the optimal resonantfrequencies of those transducers (0.44 and 0.67 MHz). The presentinvention contemplates multiple ways that are useful for creatingspecific pulse sequences, each of which are effective for modulatingcellular activity, such as stimulating neuronal activity, depending onthe desired outcome.

FIGS. 28A-F illustrates how different pulse sequences can be generatedto use ultrasound for the purposes of modulating cellular activity. InFIGS. 28A-F, each segment illustrates 3 US pulses repeated at some pulserepetition frequency (PRF). Each pulse may contain between 1 and 50,000or higher US cycles per pulse (c/p) driven by a sine wave, square wave,saw-tooth pattern, or arbitrary waveform to produce a fundamentalfrequency of 0.1 to 0.9 MHz with or without other beat and/or harmonicfrequencies ranging from 0.02 to 100 MHz. The number of US pulses per USwaveform may similarly range from 1 to 10,000,000 repeated at pulserepetition frequencies from 0.0001 to 100 MHz for total stimuluswaveforms duration from about 0.00001 sec to a continuous application tothe subject. FIG. 28A illustrates a US waveform having three pulses ofUS with each pulse having 10 US cycles produced using a 0.5 MHz sinewave and a 0.5 MHz transducer. FIG. 28B illustrates a US waveform havingthree pulses of US with each pulse having 10 US cycles produced using a0.25 MHz sine wave and a 0.5 MHz transducer. FIG. 28C illustrates USpulses used in creating the US stimulus waveform introducing multiplefrequency components in the waveform as previously discussed (FIG. 27Cabove). The waveform illustrated has three pulses of US with each pulseproduced using a 0.25 MHz square wave and a 0.5 MHz transducer. FIG.28D-E illustrate similar method outcomes for creating US stimuluswaveforms, which use multiple US frequencies to achieve modulation ofcellular activity such as neuronal activity. The US stimulus waveformsillustrated in FIG. 28D use square waves to drive the transducers verynear the transducers optimal resonant frequencies. An aspect of thepresent invention comprises use of frequencies (i.e., 0.25 MHz fartheraway from center frequency of the transducers, when the center frequencyequals 0.5 MHz) for modulating neuronal activity. Square waves, sinewaves, and/or arbitrary waves may be used to drive the transducers.

Though not wishing to be bound by a particular theory, it is currentlythought that ultrasound modulation of activity of cells or tissues mayoccur by changing the membranes of cells such as altering the activityof ion channels or ion transporter activity. Additionally, ultrasoundmodulation of activity of cells or tissues may occur by alterations ofstructures surrounding cells that then lead to alterations of cellularactivity. These concepts, and others, are encompassed in the presentinvention's contemplations of modulation of activity or activities ofcells. For example, application of ultrasound may modify the fluiddynamics of brain fluids and/or viscoelastic neuronal membranes in amanner that increases or decreases neuronal activity. Ultrasound willmodify channels to regulate ions, as well as fluid mechanics in a mannerto then directly modify the activity of cells residing in theextracellular fluids.

An aspect of the present invention comprises methods, devices andsystems for modulating cellular activity wherein an algorithm is used ina closed- or open-loop manner to evaluate feedback of cellular, tissueor organ activity, such as brain activity or hormone release or use, andthen modifying the stimulus waveform and treatment of the cells, tissue,organ or subject, based on the feedback. Use of open or closed loopfeedback devices or systems are contemplated by the present invention.

An aspect of the present invention comprises methods, devices andsystems for modulating cellular activity wherein light emitting devicesare used. For example, in a continuous wave or pulsed ultrasoundtreatment, in which certain types of imaging and/or modulation ofcellular activity is desired, a light energy emitting source, such as aLASER, LED or OLED, may be used. Ultrasound treatments may be used incombination with light treatment methods to modulate cellular activity.Such light treatment methods for modulating cellular activity maycomprise an active agent, such as pharmaceutical or exogenous geneproduct or protein or chemical compound activity present in a subject,and are known to those skilled in the art and may be incorporated in thepresent invention. For example, light emitting devices may be used forphotoactivation of compounds, or light modulation of cellular activityusing exogenous proteins such as channel rhodopsin-2, or laser ablationtherapies. Such light treatment methods may be provided concurrentlywith ultrasound waves, subsequently to ultrasound waves, or prior toultrasound wave treatment. Such light therapy or light treatment methodsmay comprise light sources, including but not limited to, LASER, LED andOLED, may use pulsed or continuous light waveforms.

The methods, devices and systems of the present invention for modulationof cellular activity by applying ultrasound waves to a subject may beused in conjunction with or as an adjunct to other treatments to thesubject. For example, low intensity or high intensity ultrasound wavesmay be used for imaging methods that generate data about the structureof a subject that are used as a map or to provide other information thatis helpful when providing ultrasound stimulus waveforms for modulationof cellular activity in that subject. Ultrasound stimulus waveforms maybe provided prior to, concurrently with, or after other treatmentregimens. For example, when a subject is undergoing brain surgery usingstandard surgical techniques, ultrasound imaging as described herein, orultrasound stimulus waveforms as described herein may be used before,during or after one or more surgical techniques. For example, in adiabetic subject who is using constant glucose monitoring devices, whichmay or may not be monitoring glucose but are referred to glucosemonitoring devices, and/or an insulin pump, may also undergo ultrasoundstimulus waveform treatment continuously, or in an intermittent, orregular treatment regimen of ultrasound. For example, a subjectundergoing pharmaceutical or chemical treatment of a condition, such ascancer, for example, a subject with a brain tumor, wherein chemotherapyis being administered, may also undergo ultrasound treatments asdescribed herein, prior to concurrently with or after each dosage ofchemotherapy or each chemotherapy cycle, or the entire chemotherapytreatment regimen.

Methods and Devices for Eliminating Brain Tumor Tissue UsingLow-Intensity, Low-Frequency Ultrasound

The present invention comprises methods of treating a subject with abrain tumor using the ultrasound devices disclosed herein. A “braintumor” is understood to be an abnormal growth of cells within the brainor inside the skull, which can be cancerous or non-cancerous (benign).It may be any intracranial tumor created by abnormal and uncontrolledcell division, normally either in the brain itself involving neurons,glial cells, astrocytes, oligodendrocytes, ependymal cells, lymphatictissue, blood vessels; in the cranial nerves (myelin-producing Schwanncells), in the brain membranes (meninges), skull, pituitary and pinealgland, or spread from cancers primarily located in other organs(metastatic tumors). Types of brain tumors include, but are not limitedto, glioma, astrocytoma, meningioma and blastoma.

For treatment of brain tumors, low-intensity, low-frequency ultrasoundincludes acoustic intensities ranging from about 0.1 mW/cm² to 100 W/cm²which are generated in the treatment region of the brain tumor tissue.Here, low-intensity, low-frequency ultrasound includes a range ofultrasound acoustic frequencies spanning from about 0.02 MHz to about10.0 MHz and ranges therein. An aspect of the invention comprisesmethods for providing low intensity ultrasound to treat the brain, andmethods for low intensity, low frequency ultrasound that will cross theskull bones to transmit ultrasound to the brain to treat the braintumor.

Ultrasound waves may be applied in a pulsed or continuous manner.Ultrasound may be focused in the brain tumor treatment region using dataacquired with medical imaging techniques such as MRI (magnetic resonanceimaging), CT (computed tomography), PET (positron emission tomography),or sonography including acoustic radiation force imaging. Alternatively,ultrasound may be applied to the brain tumor treatment region in anunfocused manner. The ultrasound can be applied to the brain tumortreatment region such that the ultrasound energy acts on brain tumorcells in a manner to induce cell-death and/or apoptosis in the braintumor cells in the absence of a deleterious increase in temperature. Theouter skin and skull may be cooled to prevent temperature damage, ifany. For example, ultrasound methods of the present invention comprisetemperature changes where the brain and/or brain tumor tissue (excludingskull) temperature does create thermal lesions in the brain, where thetemperature of the brain tissue does not exceed 44° C., or may be 35,36, 37, 38, 39, 40, 41, 42, 43, 44° C. for more than 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds at any pointduring the treatment. In one example, the temperature in the brainremains between 30° C. and 44° C. during the delivery of ultrasound tothe brain tumor treatment region. In a more specific example, thetemperature does in the brain does not exceed more than 44° C. for morethan 10 seconds during treatment.

It is known that the heating of diseased tissue induces coagulativenecrosis and thus destroys the brain tumor cells. High temperatures mayalso be generated in surrounding tissues such as vasculature, skull, ornormal brain tissue, which poses a patient being treated to unnecessarymedical risks. Thus, increasing the temperature of brain tumors bydelivering high-intensity ultrasound to destroy them may inadvertentlydamage surrounding cranial nerves (i.e., the optic nerve near the skullbase), the vasculature (near the cranium and/or skull base), or normalbrain tissue. Methods may include combination therapies where lowintensity, low frequency ultrasound methods are used to induce celldeath by activation of cells leading to activation of cell deathpathways, and application of high frequency ultrasound that provideshigh temperatures to the tissue for ablation and/or cell death due tohigh temperatures.

Ultrasound induced cell modulation or activation may comprise activationof cell death pathways including the activation of caspases and/or othercytotoxic proteins (the so called death enzymes) by ultrasound-inducedmodulation of ion channels or ion transporters, which act to regulatethe ion conductance and/or transport of calcium, potassium, chloride, orsodium across the cell membrane or across the membrane of cellularorganelles such as mitochondria, nuclei, and endoplasmic reticulum foundin brain tumor cells.

Brain tumors consist of numerous different histopathological brain celltypes. Brain tumors can be cellular identified as originating primarilyfrom glial cells including astrocytes and oligodendrocytes, neuroblasts,or other cell type found in brain tissues. The cell types making upbrain tumors have many fundamental differences, however all these celltypes can be subjected to cell death and/or apoptosis, which isactivated by various molecular cellular signaling cascades includingactivation of caspase proteins and/or other cytotoxic proteins.

It is currently believed that using low-intensity, low-frequencyultrasound reduces the risk of damaging normal brain tissue due toeither thermal effects and/or cavitational effects. Treating braintumors using low-intensity, low-frequency ultrasound reduces theprobability of increasing the temperature of either the brain tumortreatment region or the surrounding tissues (cranial nerves,vasculature, and bone). Treating brain tumors using low-intensityultrasound as described herein reduces the probability of inducingcavitation in brain tissue. The intensities currently described fortreating brain tumors by inducing coagulative necrosis through thermalmechanisms while transmitting ultrasound through the intact skull aretypically >100 W/cm². These high acoustic intensities and associatedacoustic pressures can cause undesirable thermal and/or cavitationaldamage to normal brain tissue. The probability of inducing cavitation inbrain tissue is also greatly reduced at acoustic intensities <100 W/cm²whereby peak acoustic pressures are <10 MPa.

The present invention comprises methods and devices for transmittinglow-intensity, low-frequency ultrasound in a range from about 0.1 mW/cm²to about 100 W/cm² to a brain tumor treatment region, through the skullor to exposed tissue, in a focused or unfocused manner. The intensity ofultrasound refers to the intensity generated during the delivery of asingle treatment event or ultrasound transmission event (pulsed orcontinuous wave).

The present invention comprises methods and devices for transmittinglow-intensity ultrasound to a brain tumor treatment region, through theskull or to exposed tissue, through intact or craniotomized skull, in acontinuous wave or pulsed manner ranging in duration from 0.000001seconds to 100,000 seconds during the delivery of any one ultrasoundtreatment event (a single ultrasound transmission event). An ultrasoundtreatment event may be repeated at repetition frequencies ranging fromonce about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 60, 100, 120, 150, 180, 200,240, 280, or 360 days, or more. In one example, treatment is given every30 days up to 10 KHz for a total cumulative ultrasound treatment eventtime not to exceed 94,700,000 seconds (˜3 years) using any repetitionfrequency or a combination of repetition frequencies in either single ormultiple treatment sessions.

The present invention comprises methods and devices for transmittinglow-intensity ultrasound where the ultrasound applied to a brain tumortreatment region induces peak acoustic pressures in the brain treatmenttumor region is less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 MPa,for example. In one embodiment, the peak acoustic pressure is less than10 MPa.

The present invention comprises methods and devices for transmittinglow-intensity ultrasound to a brain tumor treatment region in which theacoustic frequencies of ultrasound delivered to the brain tumor regionrepresent a single or combination of acoustic frequencies rangingminimally from about 0.05 to 10 MHz.

The present invention comprises methods and devices composed of 1 to1000 ultrasound transducer elements where plate voltages are applied tothe ultrasound transducer elements using analog or digitized waveformscomposed singly or as a combination of square, sine, saw-tooth, orarbitrary waveforms while the transducers are activated in asynchronized or phased manner. In an embodiment, the number ofultrasound transducer elements is less than 300.

Low-intensity ultrasound can be administered to a brain tumor treatmentregion whereby the action of ultrasound alone induce cell death and/orapoptosis in brain tumor cells by activating cellular molecularsignaling proteins such as caspases by modulating the ionic conductanceor transport of any of the following ions calcium, potassium, sodium,and chloride in tumor cells by regulating the activity of endogenousprotein ion channels or ion transporters on the tumor cell membrane orthe membrane of organelles found in the brain tumor cell such as thenucleus, mitochondria, or endoplasmic reticulum.

The methods disclosed herein can be administered alone, or incombination with other devices, compositions, or methods for use intreating brain tumors, other tumors, or cell death in particular cells.The present invention comprises methods and compositions which canenhance the effectiveness of the ultrasound treatment or may be used toaugment brain tumor treatment without a specific facilitation of theultrasound effect. For example, the methods of use of ultrasound taughtherein may be combined with compositions comprising recombinant proteinsor small organic molecules to induce cell death and/or apoptosis inbrain tumor cells or other tumors or cell types by activating cellularmolecular signaling proteins such as caspases by modulating the ionicconductance or transport of any of the following ions: calcium,potassium, sodium, and chloride in tumor cells by regulating theactivity of endogenous protein ion channels or ion transporters on thecell membrane or the membrane of organelles found in the brain tumorcell such as the nucleus, mitochondria, or endoplasmic reticulum.

Data from medical imaging modalities can be used to focus the ultrasonicenergy, including but not limited to, ultrasound imaging, acousticradiation force imaging, photoacoustictomography, MRI, CT, PET, or acombination thereof. These can be used before, during, or afterultrasound treatment as described herein.

Methods and Device for Treating Diabetes Using Low-Intensity,Low-Frequency Ultrasound

“Diabetes” as used herein refers to both Type I and Type II diabetesmellitus. It is currently believed that the vagus nerve plays anintegral role in regulating nutrient metabolism and physiologicalhomeostasis by innervating the stomach, intestines, pancreas, and liver.Ascending vagal afferents from these organs transmit informationregarding nutrients and food intake to the hypothalamus and other brainregions while descending vagal efferents serve to carry signals from thehypothalamus and brain back to the stomach, intestines, pancreas, andliver to govern the synthesis and secretion of metabolic factors such asinsulin, glucagon, and others. This gut-brain-gut loop is referred to asthe vago-vagal loop. In the vago-vagal loop, vagal efferent activity istriggered by nutrient consumption (fat, carbohydrate, and/or protein) asreceptors in the oropharyngeal cavity, stomach, and small intestinessense the intake of these nutrients. In response to nutrient intake andglucose buildup the activity of vagal efferents innervating the pancreasstimulate early-phase insulin release as well as serve to optimizepostprandial insulin synthesis and secretion by the β-cells ofpancreatic islets. Diabetes is a metabolic disease characterized eitherby pathologically low-levels of insulin synthesis and/or secretion inresponse to glucose buildup (Type-I Diabetes or Juvenile Diabetes) or byabnormal cellular responses to synthesized and/or secreted insulin(Type-II Diabetes or Adult-Onset Diabetes).

Electrical stimulation of vagal efferents is known to stimulate thesynthesis and secretion of insulin from the β-cells of pancreaticislets. The present invention comprises methods and devices for treatingdiabetes by stimulating the activity of vagal efferents and/or cells ingastrointestinal, nervous or related organs using low intensityultrasound. Low-intensity ultrasound may increase the activity ofnervous tissues by inducing action potentials and neurotransmitterrelease. In one embodiment, the invention comprises a method and adevice by which low-intensity ultrasound is delivered in a focused orunfocused manner to vagal afferents and or efferents in an effectivemanner to increase activity of vagal afferents and or efferents and in amanner sufficient so that insulin synthesis and/or secretion by theβ-cells of pancreatic islets in diabetics is stimulated.

The present invention comprises methods and devices for the treatment ofdiabetes by which low-intensity ultrasound is delivered in a focused orunfocused manner directly to the pancreas in a manner to stimulateproliferation (division) of pancreatic β-cells to promote insulinproduction/secretion and/or to stimulate the production/secretion ofinsulin by existing pancreatic β-cells.

In these embodiments low-intensity ultrasound is used for stimulatingthe vagus nerve to stimulate pancreatic β-cell activity or by deliveringlow-intensity ultrasound directly to the pancreas itself in a manner tostimulate β-cell activity or by combining both approaches in conjunctionwith one another. The methods are designated for deliveringlow-intensity ultrasound in a manner to promote insulin secretion bypancreatic β-cells by at a minimum increasing the calcium concentrationin the β-cells. Low-intensity ultrasound can increase the calciumconcentration in many cell types including in neurons to modulateneurotransmitter release as previously shown. Increasing calciumconcentrations and calcium activity in β-cells using low-intensityultrasound will stimulate the physiological activity of the β-cells suchthat these cells either undergo division to increase their cellulardensity to promote increased insulin production and/or secretion or suchthat the increase in calcium stimulates the production and/or secretionof insulin from existing β-cells.

Ultrasound treatments and methods of the present invention may compriseuse of other devices, such as those used for blood glucose monitoring orinsulin level detectors. Such devices may provide feedback informationto ultrasound devices so that ultrasound devices comprising transducersmay provide ultrasound, or increase or decrease ultrasound treatments inresponse to such feedback information. The present inventioncontemplates control of ultrasound transducers by feedback devices, andincludes devices capable of measuring a body parameter, transmittingthat information to an ultrasound producing device to alter or maintainthe ultrasound treatment provided, and includes applications forconditions known by those skilled in the art and those disclosed herein.Examples of aspects of methods and devices for diabetes treatment areshown in FIGS. 13-14 . In FIG. 13 , the vagus nerve, with afferents tothe brain and efferents to multiple organs, for example, thegastrointestinal tract and related organs. An ultrasound transducer,comprising from 1 to 1000 elements, for providing low intensityultrasound is shown providing ultrasound waves to efferents of the vagusnerve. The ultrasound modulates the activity of the vagus nerve and thevagus nerve stimulates the pancreas to cause synthesis of insulin,secretion or insulin or other cellular factors produced by the pancreas.Low intensity ultrasound activates the vago-vagal reflex innervating thepancreas, which in turn activates at least β-cells. The low intensityultrasound may be focused or unfocused. In FIG. 14 , low intensityultrasound is shown activating the pancreas directly. The methods ofFIG. 13 , activation of the vagal efferents and the methods of FIG. 14 ,direct activation of the pancreas, may be used in combination,sequentially or simultaneously. In FIG. 14 , an ultrasound transducer,comprising from 1 to 1000 elements, for providing low intensityultrasound is shown providing ultrasound waves to the pancreas tostimulate β-cell insulin synthesis, secretion of insulin, and/or β-celldivision and proliferation. As is known, other cell types are present inthe pancreas and these cells may or may not be stimulated, depending onthe desired treatment. The low intensity ultrasound may be focused orunfocused. The number of transducers may be 1 to 1000, and may have amixed range of frequencies. The frequencies of the transducers used maybe in a simple design, such that all frequency ranges are the same, ormay be in a complex design, in which different transducers havedifferent frequency ranges. For example, one transducer may be at 0.5MHz, the adjacent transducer at 0.7 MHz, and the adjacent transducer at0.5 MHz. The transducers may be physically arranged in any knownfunctional design, such as sequentially along a line, or spatiallyarranged in two or three dimensions to provide unique devices.

The present invention comprises methods and devices for stimulating thevagus nerve using low-intensity ultrasound (0.001 mW/cm² to 100 W/cm²;focused or unfocused) in a manner which triggers the β-cells ofpancreatic islets to increase insulin synthesis and/or insulin secretionto treat diabetes. The intensity of ultrasound refers to the intensitygenerated at the vagus nerve during the delivery of a single treatmentevent. Low-intensity ultrasound can be transmitted to the vagus nerveand/or the pancreas in a continuous wave or pulsed manner ranging induration from 0.000001 seconds to 100,000 seconds during the delivery ofany one ultrasound treatment event (a single ultrasound transmissionevent). An ultrasound treatment event may be repeated at repetitionfrequencies ranging from once about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 60,100, 120, 150, 180, 200, 240, 280, or 360 days, or more. In one example,treatment is given every 30 days up to 10 KHz for a total cumulativeultrasound treatment not to exceed the life of the patient beingtreated.

The present invention comprises methods and devices for stimulating celldivision of the β-cells of pancreatic islets by focusing low-intensityultrasound directly on the pancreas to treat diabetes. Also disclosedare methods for stimulating insulin synthesis in β-cells of pancreaticislets by focusing low-intensity ultrasound directly on the pancreas totreat diabetes. Further disclosed are methods for stimulating insulinsecretion by the β-cells of pancreatic islets by focusing low-intensityultrasound directly on the pancreas to treat diabetes.

The present invention comprises methods and devices for treatment ofdiabetes whereby the actions of ultrasound alone induce insulinsynthesis and/or secretion by increasing the calcium concentration inβ-cells of pancreatic islets. The present invention comprises methodsand devices for transmitting low-intensity ultrasound to the vagus nerveand/or pancreas for the treatment of diabetes whereby the actions ofultrasound in conjunction with recombinant proteins and/or small organicmolecules induce insulin synthesis and/or secretion by increasing thecalcium concentration in β-cells of pancreatic islets.

The present invention comprises methods and devices for transmittinglow-intensity ultrasound to the vagus nerve and/or the pancreas for thetreatment of diabetes in which the acoustic frequencies of ultrasounddelivered to the pancreas represent a single or combination of acousticfrequencies ranging minimally from 0.05 to 50 MHz. In one embodiment asingle or multiple transducers is externally coupled to the patient. Inanother embodiment a single or multiple transducers is surgicallyimplanted. In another embodiment, the activation of ultrasoundtransducers is controlled by a device for sensing glucose levels. In thepresent invention, one or more transducers may be implanted orpermanently or semi-permanently attached to a body or surface by methodsknown to those skilled in the art, such as by sutures or bolts, such astitanium or titanium alloy bolts. Such attachment elements are known tothose skilled in the art, and are not limiting to the invention.

Methods and Device for Treating Obese or Overweight Individuals UsingLow-Intensity, Low-Frequency Ultrasound

It is currently believed that the vagus nerve plays an integral role inregulating satiety, food intake, body weight, glucose levels, fatmetabolism, and nutrient homeostasis by innervating the stomach,intestines, pancreas, and liver. Along the gut-brain axis in thevago-vagal loop ascending vagal afferents from these organs transmitinformation regarding nutrients and food intake to the hypothalamus andother brain regions while descending vagal efferents serve to carrysignals from the hypothalamus and brain back to the stomach, intestines,pancreas, and liver to govern gastric/intestinal motility and function,as well as the synthesis and secretion of gastrointestinal hormones andmetabolic factors such as cholecystokinin, peptide YY, ghrelin,glucagon-like peptide, gastric inhibitory polypeptide, and enterostatinwhich all regulate to varying degrees appetite, food-intake, andsatiety.

Malfunctioning signaling in the vago-vagal loop can lead to obesitysince many of the hormonal cues eliciting meal termination do notfunction properly. Further, obesity can occur when the vagus nervecannot act in an appropriate manner to regulate fat metabolism orglucose levels. Vagal afferents undergo an increase in activity duringmeal consumption as receptors in the oropharyngeal cavity, stomach, andsmall intestines sense the intake of nutrients including fats,carbohydrates, and proteins. These afferents trigger the release oflocal satiety cues and transmit signals to various regions of thehypothalamus, which in turn leads to an increase in the activity ofvagal efferents innervating the stomach, small intestines, liver, andpancreas to regulate gastric delay, gastric motility, variousgastrointestinal hormones (described above) which trigger satiety andmeal termination, and the production and release of metabolic enzymessuch as insulin. The overall net effect of increased vagal afferent andefferent activity during nutrient consumption acts to signal satiety andregulate postprandial fat and glucose metabolism.

The present invention comprises methods and devices for deliveringfocused or unfocused low-intensity, low-frequency ultrasound to thevagus nerve or the stomach, duodenum, jejunum, liver, or pancreas, ormore than one, sequentially or concurrently, prior to, during, and/orshortly following nutrient consumption and/or food intake for thetreatment of obese or overweight individuals.

The present invention comprises methods and devices for low-intensityultrasound that are used to increase the activity of vagal nerves(afferent or efferent) to induce satiety in individuals who are obeseand/or overweight (overweight is defined herein as an individual with aBMI greater than about 25 kg/m² and obesity is an individual with a BMIgreater than about 30 kg/m²), engage in compulsive overeating or bingeeating disorders, and/or who have abnormal fat or glucose metabolismdisorders. For example, low intensity ultrasound is delivered in afocused or unfocused manner to the vagus nerve in a manner that reducesthe average caloric intake per nutrient consumption event (meal orsnack). Low intensity ultrasound is applied to the vagus nerve in amanner to increase neuronal activity beginning 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60,90, or 120 minutes prior to the time when a nutrient consumption eventincluding food intake occurs. In one example, the ultrasound is applied30 minutes prior to food or nutrient intake. The ultrasound treatmentcan last during and following nutrient consumption for 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50 51, 52, 53, 54, 55, 56, 57, 58, 59, or60, 90, or 120 minutes. In one example, ultrasound treatment iscontinued for 60 minutes after food or nutrient consumption. Lowintensity ultrasound application is applied to the vagus nerve in amanner to increase the activity of vagal afferents transmitting signalsto the arcuate nucleus and the nucleus solitary tract of the CNS for thepurposes of eliciting satiety and/or increased fat and/or glucosemetabolism.

The present invention comprises methods and devices for low-intensity,low-frequency ultrasound delivery in a focused or unfocused manner toany of anatomical regions of any of the following gastrointestinalorgans: stomach, duodenum, jejunum, liver, and pancreas. An aspect ofthe invention comprises delivery of low-intensity ultrasound to all orparts of these organs in focused or unfocused fields or to portions ofthe vagus nerve innervating them for the purposes of eliciting satietyor increasing the production or release of gastrointestinal hormones orto stimulate fat or glucose metabolism (or more than one of thosepurposes combined) for the treatment of obese or overweight individuals.In this embodiment, the actions of low-intensity ultrasound actsdirectly on the stomach, duodenum, jejunum, liver, and pancreas toinduce an upregulation of the physiological activity of any of thecellular constituents of these organs. An aspect of the inventioncomprises low-intensity ultrasound delivered in a focused or unfocusedmanner to any of the distal stomach, duodenum, and proximal jejunum in amanner which increases the release of cholecystokinin (any of itsisoforms CCK-83, CCK-58, CCK-39, CCK-33, CCK-22, and CCK-8) for thetreatment of obese or overweight individuals or those individuals whosuffer from binge eating disorders such as bulimia. In this embodimentlow-intensity ultrasound is delivered in a focused or unfocused mannerto any portion of stomach, duodenum, and proximal jejunum in a mannerwhich increases calcium activity in CCK-secreting cells to stimulate therelease of cholecystokinin from CCK-secreting cells for the treatment ofobese or overweight individuals or those individuals who suffer frombinge eating disorders.

The present invention comprises methods and devices for deliveringlow-intensity ultrasound that are wearable or personal use devices. Thedevice is worn under clothing and provides ultrasound to the appropriatelocation(s) before, during or after consumption of calories. An aspectof the invention comprises a device delivering low-intensity ultrasoundused in a clinical setting such as for those patients who need toundergo rapid weight loss programs so that the risks associated withcertain surgical procedures (GI surgery, cardiothoracic surgery, forexample) can be minimized. In an embodiment a single or multipleultrasound transducers may be surgically implanted to deliver ultrasoundwaveforms to the stomach, duodenum, jejunum, liver, and pancreas.

The present invention comprises methods and devices for stimulating thevagus nerve using low-intensity ultrasound (0.001 mW/cm² to 100 W/cm²;focused or unfocused) at the site of the tissue in a manner whichincreases the activity of vagal afferents projecting to the arcuatenucleus and/or the nucleus solitary tract for the treatment of obese oroverweight individuals. The intensity of ultrasound refers to theintensity generated during the delivery of a single treatment event.

The present invention comprises methods and devices for treating obeseor overweight individuals by transmitting low-intensity ultrasound in afocused or unfocused manner to the vagus nerve or any portion of one ormore of the stomach, duodenum, jejunum, pancreas, and liver in acontinuous wave or pulsed manner ranging in duration from 0.000001seconds to 100,000 seconds during the delivery of any one ultrasoundtreatment event (a single ultrasound transmission event). An ultrasoundtreatment event may be repeated at repetition frequencies ranging fromonce about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 60, 100, 120, 150, 180, 200,240, 280, or 360 days, or more. In one example, treatment is given every30 days up to 10 KHz for a total cumulative ultrasound treatment not toexceed the life of the patient being treated.

The present invention comprises methods and devices for treating obeseor overweight individuals where low-intensity ultrasound is applied in afocused or unfocused manner to the vagus nerve or any portion of thestomach, duodenum, jejunum, pancreas, and liver in a manner whichincreases their physiological activity in a manner to increase satietyand/or fat and/or glucose metabolism.

The present invention comprises methods and devices for transmittingfocused and/or unfocused low-intensity ultrasound to the vagus nerveand/or any portion of any of the stomach, duodenum, jejunum, liver, orpancreas for the treatment of obese or overweight individuals in whichthe acoustic frequencies of ultrasound delivered represent a single orcombination of acoustic frequencies ranging minimally from 0.05 to 50MHz.

The present invention comprises methods and devices for transmittingfocused and/or unfocused low-intensity ultrasound to the vagus nerveand/or any portion of any of the stomach, duodenum, jejunum, liver, orpancreas for the treatment of obese or overweight individuals in which adevice may be composed of 1 to 1000 ultrasound transducer elements whereplate voltages are applied to the ultrasound transducer elements usinganalog or digitized waveforms composed singly or as a combination ofsquare, sine, saw-tooth, or arbitrary waveforms, and wherein thetransducers may be activated in a synchronized or phased manner.

The present invention comprises methods and devices for transmittingfocused and/or unfocused low-intensity ultrasound to the vagus nerveand/or any portion of any of the stomach, duodenum, jejunum, liver, orpancreas for the treatment of obese or overweight individuals in which adevice delivers ultrasound in at least some portion of the treatment ina focused manner, and delivery of focused ultrasound is uses dataacquired by medical imaging modalities such as ultrasound imaging, MRI,PET, or others known in the art.

FIGS. 15 and 16 show examples of methods and devices for treatment ofobesity. In FIG. 15 , ultrasound waves are provided by one or moretransducers, such as piezoelectric steering devices, and or by one ormore scanning transducer arrays. The vagus nerve efferents and afferentsand some portion of one or more gastrointestinal organs may be swept bya low frequency, low intensity ultrasound field. Such a system may beused to deliver a swept, unfocused field of low intensity ultrasound tomultiple GI organs and nerves to increase the physiological activity ofthe vagus nerve and at least a portion of one or more of the pancreas,the stomach, the duodenum, the jejunum and the liver. Not all the organsare shown in the figures. The number of transducers may be 1 to 1000,and may have a mixed range of frequencies. The frequencies of thetransducers used may be in a simple design, such that all frequencyranges are the same, or may be in a complex design, in which differenttransducers have different frequency ranges. For example, one transducermay be at 0.5 MHz, the adjacent transducer at 0.7 MHz, and the adjacenttransducer at 0.5 MHz. The transducers may be physically arranged in anyknown functional design, such as sequentially along a line, or spatiallyarranged in two or three dimensions to provide unique devices.

FIG. 16 shows methods and devices for direct stimulation with lowintensity ultrasound of at least a portion of one or moregastrointestinal organs such as the stomach, duodenum and proximaljejunum. Direct stimulation of at least a portion of one or moregastrointestinal organs may be used in conjunction with stimulation ofthe vagus nerve afferents or efferents, which may comprise sequential orsimultaneous activation by low frequency, low intensity ultrasound. Inthis example, from 1 to 300 ultrasound transducers are used to modulatethe cells of at least a portion of one or more gastrointestinal organssuch as the stomach, duodenum and proximal jejunum. An outcome of suchstimulation may result in stimulation of CCK signaling.

The present invention comprises methods and devices for stimulatingnerves and or organs to decrease fat metabolism and to increase bodyweight. For example, vagal efferents may be stimulated as describedherein, and/or stimulation of hypothalamic regions of the brain maycause weight gain, or inhibit fat metabolism. Such use of ultrasound maybe useful in methods of treatment for subjects with anorexia, bulimia,cachexia, or conditions where weight gain is desired, such as duringchemotherapy (cancer) treatment or AIDS.

Such weight gain or weight loss methods may be used in conjunction withtreatments for conditions such as depression, recovery from surgery orinjury, or other conditions wherein weight control is an adjunct issueof the primary condition.

Method and Device Using Low-Intensity, Low-Frequency Ultrasound forReducing Secondary Brain Damage Following Traumatic Brain Injury ofConcussive Event

Traumatic brain injury is one of the leading causes of death andmorbidity in North America and is a rising global healthcare problemworldwide. Among the patients who die as a result of TBI, approximately90% die within 48 hours of the primary injury. Over recent years it hasbeen shown that the pathophysiological events associated with TBI orconcussive head trauma are delayed and progressive in nature. Thesedelayed and progressive pathophysiological events following head traumaoften induce “secondary injury”. Secondary injury following head traumainvolves a host of molecular cellular signaling cascades and responsesin glial cells and neurons including toxicity due to reactive oxygenspecies (free radicals), glutamate-mediated excitotoxicity, hypoxia,mechanical damage due to high intracranial pressure, excessive calciuminflux, disrupted ionic homeostasis, release of pro-inflammatorycytokines, and reactive gliosis. Secondary injuries also occur followingmild TBI or concussive head trauma even when the injury may seem “mild”or when the injury does not induce loss of consciousness. Secondaryinjuries also occur in white matter following traumatic events such aswhiplash and can produce diffuse axonal injury (DAI). Secondary injuryin both gray and white matter often results from glutamate-mediatedexcitotoxicity, disrupted calcium homeostasis, and activation of celldeath pathways. Preventing the deleterious consequences of thesesecondary injuries by dampening the effects the molecular cellularsignaling cascades triggered following TBI or DAI can lead to improvedfunctional outcomes during recovery processes, as well as increasedsurvival rates by minimizing the impact of the delayed sequelae andprogressive pathophysiology.

Methods of treatment comprising early medical attention duringpre-hospital care and other acute medical interventions may reduce thedamage associated with secondary injury following TBI or DAI. Earlymedical attention in reducing intracerebral pressure has been attributedto saving many lives and reducing death related to secondary injuryfollowing TBI. Several small molecules and biological factors exertingneuroprotective effects have been examined and are thought to protectthe brain and nervous system from damage due to secondary injuryfollowing TBI or DAI. These factors include anti-inflammatory agents,glutamate receptor modulators (AMPA and NMDA glutamate receptorsubtype), neurotrophic factors such as brain-derived neurotrophic factor(BDNF), apoptotic inhibitors, ion channel modulators, nitric oxidemodulators, and a host of other pharmacological agents. The inventionhere describes a method and device for delivering low-intensity,low-frequency ultrasound in a focused or unfocused manner to the brainor nervous system such that the ultrasonic energy inducesneuroprotection to mitigate some of the pathophysiological consequencesof secondary injury following TBI or DAI.

An aspect of the invention comprises providing low-intensity,low-frequency (0.1 to 10 MHz) ultrasound which penetrates the intactskull and is transmitted into the brain in a focused and/or unfocusedmanner. Such low-intensity, low-frequency ultrasound methods triggerbioeffects on numerous cellular molecular cascades in a manner toupregulate the production and/or secretion of one or more factors toexert neuroprotection, promote neuronal plasticity, and increaseneuronal survival. Low-intensity ultrasound can increase transforminggrowth factor beta (TGF-β), insulin-like growth factor receptor-I (IGF),interleukin-8 (IL), basic fibroblast growth factor (bFGF), vascularendothelial growth factor (VEGF), and nitric oxide signaling events inmany different cell types. Besides its angiogenic activities, bFGFmodulates synaptic transmission, is a potent regulator of neuronalsurvival and VEGF, TGF-β, and bFGF are also neuroprotective againstinjury and neurodegeneration. NF-κB is known to regulate neuronalsurvival and plasticity and the PI3K-Akt signaling pathway is capable ofblocking cell death and promoting cell survival of many neuronal celltypes. Low-intensity, low frequency ultrasound activates Akt/NF-κB andPI3K/Akt signaling pathway in many cell types. Low-intensity,low-frequency ultrasound increases the synthesis and release of nitricoxide in many cell types, as well as increasing the activity of nitricoxide synthase. Nitric oxide (NO) can mediate neuroprotection, celldeath, and neurogenesis following traumatic brain injury. BDNF is aneurotrophic factor, which has cell survival and neuroprotective effectsand in addition is a potent regulator of ion channel modulation, as wellas neurotransmitter receptors throughout the brain. Here, TBI refers tosevere or mild traumatic brain injury where a head trauma has induced aloss of consciousness or not.

The present invention comprises methods and devices for usinglow-intensity ultrasound (0.001 mW/cm² to 100 W/cm²; focused orunfocused) at the site of the brain tissue for increasing the signalingactivity of neuroprotective molecules (including any of BDNF, NO, NOS,VEGF, TGF-β, bFGF, NF-κB, or PI3K-Akt) to protect the brain (reduce celldeath) of any individual from the deleterious consequences of secondaryinjury following a TBI. The intensity of ultrasound refers to theintensity generated during the delivery of a single treatment event.

The present invention comprises methods and devices for reducing thedeleterious consequences of secondary injury following a TBI bytransmitting low-intensity ultrasound in a focused and/or unfocusedmanner to the brain or any portion thereof, where the ultrasound isapplied in a continuous wave or pulsed manner ranging in duration from0.000001 seconds to 1,000,000 seconds during the delivery of any oneultrasound treatment event (a single ultrasound transmission event.) Anultrasound treatment event may be repeated at repetition frequenciesranging from once about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 60, 100, 120,150, 180, 200, 240, 280, or 360 days, or more. In one example, treatmentis given every 30 days up to 10 KHz for a total cumulative ultrasoundtreatment not to exceed the life of the patient being treated.

The present invention comprises methods and devices for reducing thedeleterious consequences of secondary injury following a TBI bytransmitting low-intensity ultrasound in a focused or unfocused mannerto the brain or any portion thereof in a manner which modulates calciumactivity. Throughout, the term “modulates calcium activity” means thatcalcium levels in a cell are increased or decreased in comparison tobasal levels or to a control.

The methods disclosed herein can reduce the deleterious consequences ofsecondary injury following a TBI by transmitting low-intensityultrasound in a focused or unfocused manner to the brain or any portionthereof at any time beginning immediately after TBI up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 days post-injury. The treatment cancontinue at regular intervals as needed, such as daily, weekly, ormonthly. A device of the present invention can be administered by anemergency first responder (i.e., paramedic, EMT), in any pre-hospitalfirst-aid, or during acute medical care (hospital ER). It can also beadministered during routine care by a physician, or can beself-administered by a patient. The acoustic frequencies of theultrasound delivered can represent a single or combination of acousticfrequencies ranging minimally from 0.05 to 50 MHz.

The present invention comprises methods and devices for reducing thedeleterious consequences of secondary injury following a TBI or otherbrain injury by transmitting low frequency, low intensity ultrasound ina focused or unfocused manner to at least a portion of the brain whereina device comprises from 1 to 1000 ultrasound transducer elements andplate voltages are applied to the transducer elements using analog oranalog or digitized waveforms composed singly or as a combination ofsquare, sine, saw-tooth, or arbitrary waveforms while the transducersare activated in a synchronized or phased manner. In one embodiment thenumber of ultrasound transducer elements is less than 300.

The present invention comprises methods and devices for reducing thedeleterious consequences of secondary injury following a TBI or otherbrain injury by transmitting low frequency, low intensity ultrasound ina focused or unfocused manner to at least a portion of the brain inwhich a device delivers ultrasound in at least some portion of thetreatment in a focused manner, and delivery of focused ultrasound usesdata acquired by medical imaging modalities such as ultrasoundimagining, MRI, PET, or others known in the art. In one embodiment theultrasound is not focused. In an embodiment, the ultrasound is focused.In an embodiment, the ultrasound may be delivered in a combination offocused and unfocused manners.

FIG. 17 is illustrative of methods and devices for rapid treatment ofbrain injury with low frequency, low intensity ultrasound. A device maycomprise one transducer to provide low frequency, low intensityultrasound or a device may comprise multiple transducers, such as from 2to 300 transducers spatially arranged to treat the head. Suchtransducers may be acoustically coupled to the head by elements known inthe art. Acoustic coupling may comprise air, water-based media,including, but not limited to, gel, water or fluid filled items such assponges or other polymer materials, or other materials which have lowacoustic attenuation coefficients. These are useful where the skull isfairly intact. A device may also comprise piezoelectric drives ormicromotor drives or other components used to translate the spatialposition of transducers in order to target particular regions to bemodulated by ultrasound. Such devices may be employed in a mannersimilar to that of AEDs used for rapid cardiac response. The ultrasounddevices may be deployed in first responder sites, such as ambulances orhelicopters, or on site in buildings such as office towers or airports.Devices may also be deployed in emergency rooms for treatment duringtriage and acute early intervention. Devices may be used with consciousor unconscious individuals. The delivery of low frequency, low intensityultrasound is beneficial when applied as soon as possible to the brainof a victim of TBI to provide modulation of cellular activities in thebrain and stimulate neuroprotective cellular molecular signalingcascades including, but not limited to, BDNF signaling. Cell death andother secondary injuries are reduced.

Methods and Device Using Low-Intensity, Low-Frequency Ultrasound toTreat Other Neurological Diseases and Conditions

The methods and devices of the present invention may be used with organsystems to treat acute and chronic pathologies by modulating cellularactivities, including neural and non-neural cells, by providing lowfrequency, low intensity ultrasound. An effect of providing lowfrequency, low intensity ultrasound is to stimulate cell signalingpathways. Thus, the methods and devices of the present invention areuseful in treatments for many organs, organ systems, nerve systems, andpathological conditions found in humans, animals, plants and otherliving organisms. For example, the present invention comprises methodsand devices for treating cardiac arrhythmia in a subject comprising:acoustically coupling a low intensity ultrasound transducer device tothe subject; and driving the ultrasound transducer to form stimuluswaveform with an intensity in a range of 0.001 to 900 mW/cm² at the siteof the tissue. Testing with other procedures or patient response mayindicate the effectiveness of the treatment, and determine repeating UStreatment or altering of US treatment.

The present invention comprises methods and devices for treating asubject in a minimally conscious state, a coma, or a vegetative state,the method comprising acoustically coupling a low intensity ultrasoundtransducer device to the subject; and driving the ultrasound transducerto form stimulus waveform with an intensity in a range of 0.001 mW/cm²to 900 milliWatts per square centimeter at the site of the tissue.Testing with other procedures or patient response may indicate theeffectiveness of the treatment, and determine repeating US treatment oraltering of US treatment.

The present invention comprises methods and devices for treating asubject with locked-in syndrome comprising acoustically coupling a lowintensity ultrasound transducer device to the subject; and driving theultrasound transducer to form stimulus waveform with an intensity in arange of 0.001 to 900 milliWatts per square centimeter (mW/cm²) at thesite of the tissue. Testing with other procedures or patient responsemay indicate the effectiveness of the treatment, and determine repeatingUS treatment or altering of US treatment.

The present invention comprises methods and devices for treating asubject with a spinal cord injury comprising acoustically coupling a lowintensity ultrasound transducer device to the subject; and driving theultrasound transducer to form stimulus waveform with an intensity in arange of 0.001 to 900 milliWatts per square centimeter (mW/cm²) at thesite of the tissue. Testing with other procedures or patient responsemay indicate the effectiveness of the treatment, and determine repeatingUS treatment or altering of US treatment.

The present invention comprises methods and devices for treating asubject with back pain comprising acoustically coupling a low intensityultrasound transducer device to the subject; and driving the ultrasoundtransducer to form stimulus waveform with an intensity in a range of0.001 to 900 milliWatts per square centimeter (mW/cm²) at the site ifthe tissue. Testing with other procedures or patient response mayindicate the effectiveness of the treatment, and determine repeating UStreatment or altering of US treatment.

The present invention comprises methods and devices for treating asubject with migraine headaches comprising acoustically coupling a lowintensity ultrasound transducer device to the subject; and driving theultrasound transducer to form stimulus waveform with an intensity in arange of 0.001 to 900 milliWatts per square centimeter (mW/cm²) at thesite of the tissue. Testing with other procedures or patient responsemay indicate the effectiveness of the treatment, and determine repeatingUS treatment or altering of US treatment.

The present invention comprises methods and devices for treatingParkinson's disease or essential tremor in a subject comprisingacoustically coupling a low intensity ultrasound transducer device tothe subject; and driving the ultrasound transducer to form stimuluswaveform with an intensity in a range of 0.001 to 900 milliWatts persquare centimeter (mW/cm²) at the site of the tissue or neuronal circuitto be treated. Testing with other procedures or patient response mayindicate the effectiveness of the treatment, and determine repeating UStreatment or altering of US treatment.

Methods and Device Using Low-Intensity, Low-Frequency Ultrasound forFunctional Brain Mapping

The present invention comprises methods and devices for conductingnon-invasive functional brain mapping using low intensity ultrasound ina subject comprising acoustically coupling a low intensity ultrasoundtransducer device to the subject; and driving the ultrasound transducerto form stimulus waveform with an intensity in a range of 0.001 to 900milliWatts per square centimeter (mW/cm²) at the site of the tissue.Devices for reading neuronal activity, such as MEG, MRI, EEG, fMRI, PET,acoustic radiation force imaging, photoacoustic tomography and otherscan be used in conjunction or combination with providing ultrasoundwaves such that the neurosignal resulting from the ultrasound isrecorded and/or monitored or mapped. Such measurements may occurin >0.0000001 second from the onset of the ultrasound stimulationwaveform and last during and following the waveform for a period of upto about 1000 seconds.

The present invention comprises methods and devices for targetedneuromodulation comprising utilizing imaging data in a subject to directthe ultrasound treatment of the subject, wherein the ultrasound isprovided by acoustically coupling a low intensity ultrasound transducerdevice to the subject; and driving the ultrasound transducer to formstimulus waveform with an intensity in a range of 0.001 to 900milliWatts per square centimeter (mW/cm²) at the site of the tissue. Theimaging data may be derived from devices that provide sonography, MRI,PET, photoaccoustic tomography, tissue pulsatility imaging, acousticradiation force imaging, vibrography or other methods, and such devicesmay be combined with ultrasound devices of the present invention toprovide low frequency, low intensity ultrasound treatments focused bydirection from the imaging data. Ultrasound data imaging may compriseuse of high intensity ultrasound, such as MR-thermometry, for datarelating to location of treatment sites, and such high intensityultrasound for imaging may comprise ultrasound with up to 1000 W/cm².The present invention contemplates use of ultrasound imaging techniqueswherein data is generated using high or low intensity ultrasound, andsuch imaging may be used with the modulation of cellular activity, suchas neuromodulation, disclosed herein.

In the methods of the present invention, imaging may be accomplished byacoustic radiation force imaging (ARFI) or tissue pulsatile imaging. Useof such imaging techniques may replace the use of MRI. Ultrasoundimaging may helpful in visualizing where the ultrasound is being steeredthrough the skull. In an aspect of the invention using ARFI or tissuepulsatile imaging, the frequency of ultrasound may range from 0.01 to 5MHz. Methods of the present invention comprise conducting ultrasoundimaging of a portion of a subject, and then using the imaging datagenerated, to act as a guide to the location where ultrasound treatment,such as an ultrasound stimulus waveform, is to be provided. For example,the present invention comprises methods, systems and devices for usinglow intensity ultrasound to acquire a map of the brain vasculature foruse as a guide to provide information for conducting ultrasoundneuromodulation or other tissue modulation. For example, ultrasoundimaging data, or other imaging data, may be used to map metabolicactivity in a subject and the data regarding the location of themetabolic activity acts as a guide to the location where ultrasoundtreatment, such as an ultrasound stimulus waveform, is to be provided.

Methods and Device Using Low-Intensity, Low-Frequency Ultrasound forNeuromodulation

The present invention comprises methods and devices for modulatingneuronal cellular activity in a subject comprising transcraniallytransmitting sets of pulsed ultrasound waveforms. Methods for modulatingneuronal cellular activity comprise acoustically coupling an ultrasoundtransducer to an external surface of a subject, and driving theultrasound transducer to form stimulus waveform with an intensity belowabout 900 milliWatts per square centimeter (mW/cm²) and an ultrasoundfrequency below about 0.9 MegaHertz (MHz) at the site of the tissue. Thefrequencies comprise single components, multiple components, or acombination thereof. An aspect of the present invention comprisesultrasound waveforms having and ultrasound frequency ranging from about0.25 to about 0.50 MHz. In an aspect of the present invention, theultrasound waveforms act in a non-thermal fashion without causingsignificant heating of the tissue being treated.

The present invention comprises methods and devices for modulatingneuronal cellular activity in a subject comprising transcraniallytransmitting sets of pulsed ultrasound waveforms, wherein the waveformcomprises a plurality of single pulses. An aspect of the presentinvention comprises single pulses that have a pulse duration rangingfrom about 0.16 to about 0.57 msec. An aspect of the present inventioncomprises single pulses that are repeated at a pulse repetitionfrequency ranging from about 1.2 to about 3.0 KHz to producespatial-peak temporal-average intensities ranging from about 21 to about163 mW/cm². In an aspect of the present invention, single pulsescomprise between about 80 and about 225 acoustic cycles. Pulses can begenerated by brief bursts of waves. Waves may be one or more of knownwave, including but not limited to, sine, square, saw tooth andtriangle. The ultrasound waves may be focused to provide action at aparticular site in or on the subject, or the waves may be unfocused andprovide action at multiple sites. The waves may be continuous or pulsed,depending on the desired application. The frequency or intensity may beuniform throughout a treatment period, or may alternate or sweep fromone number to another, and back to the original number. Those skilled inthe art are able to determine such parameters for the desiredapplication. Examples are disclosed herein.

An aspect of the present invention comprises methods and devices formodulating neuronal cellular activity in a subject comprisingtranscranially transmitting sets of pulsed ultrasound waveforms, whereinthe duration of the transcranial transmission ranges from about 26 toabout 333 msec.

An aspect of the present invention comprises methods and devices formodulating neuronal cellular activity in a subject comprisingtranscranially transmitting sets of pulsed ultrasound waveforms, whereinneuronal cellular activity is modulated through ion channel or iontransporter activity. In an aspect of the present invention, the ionchannel or ion transporter regulates activity of calcium, potassium,chloride, or sodium. In an aspect of the present invention, the neuronalcellular activity relates to the secretion of signaling molecules, theproliferation of cells, the differentiation of cells, the modulation ofprotein transcription, the modulation of protein translation, or acombination thereof.

The present invention comprises methods and devices for modulatingneuronal cellular activity in a subject comprising transcraniallytransmitting sets of pulsed ultrasound waveforms, wherein the ultrasoundtransducer comprises piezoelectric transducers, composite transducers,CMUTs, or a combination thereof. In an aspect of the invention, theultrasound transducer can be implanted onto the subject's skull ormounted onto the subject's skull. For example, in an aspect, a CMUT isimplanted on the skull. In another aspect, a CMUT can also be mountedonto the subject's skull in a chronically wearable device.

The present invention comprises methods and devices for modulatingneuronal cellular activity in a subject comprising transcraniallytransmitting sets of pulsed ultrasound waveforms, wherein the ultrasoundtransducer comprises up to about 1000 elements. In an aspect of thepresent invention, the number of elements is 1, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, or 1000 elements. In an aspect of thepresent invention, the number of elements ranges from about 1 to about299.

The present invention comprises methods and devices for modulatingneuronal cellular activity in a subject comprising transcraniallytransmitting sets of pulsed ultrasound waveforms, wherein the method formodulating neuronal cellular activity is used in conjunction with EEG,MEG, MRI, PET, acoustic radiation force imaging, photoacoustictomography, or a combination thereof. In an aspect of the presentinvention, the method for modulating neuronal cellular activity furthercomprising using an algorithm in a closed- or open-loop manner toevaluate feedback of brain activity and modifying the stimulus waveformbased on that feedback.

The present invention comprises methods and devices for using acousticpressure of ultrasound to induce fluid mechanical actions in nervoustissues, brain, or brain circuits in order to modulate neuronalactivity.

The present invention comprises transcranial ultrasound waveform formodulating cellular activity. In an aspect of the present invention, thetranscranial ultrasound waveform is used in conjunction with anacoustically coupled ultrasound transducer comprising an intensity belowabout 900 milliWatts per square centimeter (mW/cm²) and an ultrasoundfrequency below about 0.9 MegaHertz (MHz), preferably ranging from about0.25 to about 0.50 MHz. In an aspect of the invention, the ultrasoundwaveform comprises a plurality of single pulses. A single pulse can havea pulse duration ranging from about 0.16 to about 0.57 msec. Singlepulses can be repeated at a pulse repetition frequency ranging fromabout 1.2 to about 3.0 KHz to produce spatial-peak temporal-averageintensities ranging from about 21 to about 163 mW/cm². A single pulsecomprises between about 80 and about 225 acoustic cycles. In an aspectof the present invention, the duration of the transcranial transmissionranges from about from about 26 to about 333 msec. In an aspect of thepresent invention, driving the acoustically coupled ultrasoundtransducer comprises piezoelectric transducers, composite transducers,CMUTs, or a combination thereof.

The present invention comprises methods and devices wherein a computeris used. For example, FIG. 12 is a block diagram that illustrates acomputer system 1300 upon which an embodiment of the invention may beimplemented. Computer system 1300 includes a communication mechanismsuch as a bus 1310 for passing information between other internal andexternal components of the computer system 1300. Information isrepresented as physical signals of a measurable phenomenon, typicallyelectric voltages, but including, in other embodiments, such phenomenaas magnetic, electromagnetic, pressure, chemical, molecular atomic andquantum interactions. For example, north and south magnetic fields, or azero and non-zero electric voltage, represent two states (0, 1) of abinary digit (bit). A sequence of binary digits constitutes digital datathat is used to represent a number or code for a character. A bus 1310includes many parallel conductors of information so that information istransferred quickly among devices coupled to the bus 1310. One or moreprocessors 1302 for processing information are coupled with the bus1310. A processor 1302 performs a set of operations on information. Theset of operations include bringing information in from the bus 1310 andplacing information on the bus 1310. The set of operations alsotypically include comparing two or more units of information, shiftingpositions of units of information, and combining two or more units ofinformation, such as by addition or multiplication. A sequence ofoperations to be executed by the processor 1302 constitutes computerinstructions.

Computer system 1300 also includes a memory 1304 coupled to bus 1310.The memory 1304, such as a random access memory (RAM) or other dynamicstorage device, stores information including computer instructions.Dynamic memory allows information stored therein to be changed by thecomputer system 1300. RAM allows a unit of information stored at alocation called a memory address to be stored and retrievedindependently of information at neighboring addresses. The memory 1304is also used by the processor 1302 to store temporary values duringexecution of computer instructions. The computer system 1300 alsoincludes a read only memory (ROM) 1306 or other static storage devicecoupled to the bus 1310 for storing static information, includinginstructions, that is not changed by the computer system 1300. Alsocoupled to bus 1310 is a non-volatile (persistent) storage device 1308,such as a magnetic disk or optical disk, for storing information,including instructions, that persists even when the computer system 1300is turned off or otherwise loses power.

Information, including instructions, is provided to the bus 1310 for useby the processor from an external input device 1312, such as a keyboardcontaining alphanumeric keys operated by a human user, or a sensor. Asensor detects conditions in its vicinity and transforms thosedetections into signals compatible with the signals used to representinformation in computer system 1300. Other external devices coupled tobus 1310, used primarily for interacting with humans, include a displaydevice 1314, such as a cathode ray tube (CRT) or a liquid crystaldisplay (LCD) or a display comprised of light emitting diodes (LED) ororganic light emitting diodes (OLED), for presenting images, and apointing device 1316, such as a mouse or a trackball or cursor directionkeys, for controlling a position of a small cursor image presented onthe display 1314 and issuing commands associated with graphical elementspresented on the display 1314.

In the illustrated embodiment, special purpose hardware, such as anapplication specific integrated circuit (IC) 1320, is coupled to bus1310. The special purpose hardware is configured to perform operationsnot performed by processor 1302 quickly enough for special purposes.Examples of application specific ICs include graphics accelerator cardsfor generating images for display 1314, cryptographic boards forencrypting and decrypting messages sent over a network, speechrecognition, and interfaces to special external devices, such as roboticarms and medical scanning equipment that repeatedly perform some complexsequence of operations that are more efficiently implemented inhardware.

Computer system 1300 also includes one or more instances of acommunications interface 1370 coupled to bus 1310. Communicationinterface 1370 provides a two-way communication coupling to a variety ofexternal devices that operate with their own processors, such asprinters, scanners and external disks. In general the coupling is with anetwork link 1378 that is connected to a local network 1380 to which avariety of external devices with their own processors are connected. Forexample, communication interface 1370 may be a parallel port or a serialport or a universal serial bus (USB) port on a personal computer. Insome embodiments, communications interface 1370 is an integratedservices digital network (ISDN) card or a digital subscriber line (DSL)card or a telephone modem that provides an information communicationconnection to a corresponding type of telephone line. In someembodiments, a communication interface 1370 is a cable modem thatconverts signals on bus 1310 into signals for a communication connectionover a coaxial cable or into optical signals for a communicationconnection over a fiber optic cable. As another example, communicationsinterface 1370 may be a local area network (LAN) card to provide a datacommunication connection to a compatible LAN, such as Ethernet. Wirelesslinks may also be implemented. Carrier waves, such as acoustic waves andelectromagnetic waves, including radio, optical and infrared wavestravel through space without wires or cables. Signals include man-madevariations in amplitude, frequency, phase, polarization or otherphysical properties of carrier waves. For wireless links, thecommunications interface 1370 sends and receives electrical, acoustic orelectromagnetic signals, including infrared and optical signals thatcarry information streams, such as digital data.

The term computer-readable medium is used herein to refer to any mediumthat participates in providing information to processor 1302, includinginstructions for execution. Such a medium may take many forms,including, but not limited to, non-volatile media, volatile media andtransmission media. Non-volatile media include, for example, optical ormagnetic disks, such as storage device 1308. Volatile media include, forexample, dynamic memory 1304. Transmission media include, for example,coaxial cables, copper wire, fiber optic cables, and waves that travelthrough space without wires or cables, such as acoustic waves andelectromagnetic waves, including radio, optical and infrared waves.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, a hard disk, a magnetic tape, or any othermagnetic medium, a compact disk ROM (CD-ROM), a digital video disk (DVD)or any other optical medium, punch cards, paper tape, or any otherphysical medium with patterns of holes, a RAM, a programmable ROM(PROM), an erasable PROM (EPROM), a FLASH-EPROM, or any other memorychip or cartridge, a carrier wave, or any other medium from which acomputer can read.

Network link 1378 typically provides information communication throughone or more networks to other devices that use or process theinformation. For example, network link 1378 may provide a connectionthrough local network 1380 to a host computer 1382 or to equipment 1384operated by an Internet Service Provider (ISP). ISP equipment 1384 inturn provides data communication services through the public, world-widepacket-switching communication network of networks now commonly referredto as the Internet 1390. A computer called a server 1392 connected tothe Internet provides a service in response to information received overthe Internet. For example, server 1392 provides information representingvideo data for presentation at display 1314.

The invention is related to the use of computer system 1300 forimplementing the techniques described herein. According to oneembodiment of the invention, those techniques are performed by computersystem 1300 in response to processor 1302 executing one or moresequences of one or more instructions contained in memory 1304. Suchinstructions, also called software and program code, may be read intomemory 1304 from another computer-readable medium such as storage device1308. Execution of the sequences of instructions contained in memory1304 causes processor 1302 to perform the method steps described herein.In alternative embodiments, hardware, such as application specificintegrated circuit 1320, may be used in place of or in combination withsoftware to implement the invention. Thus, embodiments of the inventionare not limited to any specific combination of hardware and software.

The signals transmitted over network link 1378 and other networksthrough communications interface 1370, carry information to and fromcomputer system 1300. Computer system 1300 can send and receiveinformation, including program code, through the networks 1380, 1390among others, through network link 1378 and communications interface1370. In an example using the Internet 1390, a server 1392 transmitsprogram code for a particular application, requested by a message sentfrom computer 1300, through Internet 1390, ISP equipment 1384, localnetwork 1380 and communications interface 1370. The received code may beexecuted by processor 1302 as it is received, or may be stored instorage device 1308 or other non-volatile storage for later execution,or both. In this manner, computer system 1300 may obtain applicationprogram code in the form of a signal on a carrier wave.

Various forms of computer readable media may be involved in carrying oneor more sequence of instructions or data or both to processor 1302 forexecution. For example, instructions and data may initially be carriedon a magnetic disk of a remote computer such as host 1382. The remotecomputer loads the instructions and data into its dynamic memory andsends the instructions and data over a telephone line using a modem. Amodem local to the computer system 1300 receives the instructions anddata on a telephone line and uses an infra-red transmitter to convertthe instructions and data to a signal on an infra-red a carrier waveserving as the network link 1378. An infrared detector serving ascommunications interface 1370 receives the instructions and data carriedin the infrared signal and places information representing theinstructions and data onto bus 1310. Bus 1310 carries the information tomemory 1304 from which processor 1302 retrieves and executes theinstructions using some of the data sent with the instructions. Theinstructions and data received in memory 1304 may optionally be storedon storage device 1308, either before or after execution by theprocessor 1302.

The present invention discloses methods, systems and devices formodulating cellular activity in a subject. In general, the presentinvention comprises acoustically coupling at least one component forgenerating ultrasound waves to an external surface of a subject, anddriving at least one component for generating ultrasound waves to format least one ultrasound stimulus waveform, wherein the stimulus waveformcomprises one or more frequencies, with an intensity in a range fromabout 0.0001 to about 900 mW/cm² and a frequency in a range from about0.02 to about 1.0 MHz, at the site of the cells to be modulated. Theultrasound stimulus waveform may comprise at least an ultrasoundfrequency ranging from about 0.10 to about 0.90 MHz. The ultrasoundstimulus waveform may comprise single- or multiple-componentfrequencies. The duration of one or more ultrasound stimulus waveformsmay range from about from about 0.01 to about 10000 msec. The ultrasoundstimulus waveform may comprise a plurality of single pulses, wherein asingle pulse has a pulse duration ranging from about 0.001 to about10000 msec. In an aspect of the invention, single pulses may be repeatedat a pulse repetition frequency ranging from about 0.001 to about 100KHz to produce spatial-peak temporal-average intensities ranging fromabout 21 to about 500 mW/cm². In an aspect of the present invention, asingle pulse may comprise between about 1 and about 50,000 acousticcycles. In the presently disclosed methods, a pulse may be generated bybrief bursts of square waves, sine waves, saw-tooth waveforms, sweepingwaveforms, or arbitrary waveforms, or combinations of one or morewaveforms. The waveforms may be focused or not focused. The method maybe repeated. The components for generating ultrasound, such asultrasound transducer or its elements, are driven using analog ordigitized waveforms. Ultrasound transducer elements may be driven usingindividual waveforms or a combination of square, sine, saw-tooth, orarbitrary waveforms.

Methods for modulating cellular activity in a subject may furthercomprise detecting modulated cellular activity in cells. Modulatedcellular activity includes but is not limited to changes in (i) the ionchannel activity, (ii) the ion transporter activity, (iii) the secretionof signaling molecules, (iv) the proliferation of the cells, (v) thedifferentiation of the cells, (vi) the protein transcription of thecells, (vii) the protein translation of cells, (viii) the proteinphosphorylation of the cells, (ix) the protein structures in the cells,or a combination thereof.

In the present invention, at least one component for generatingultrasound waves comprises an ultrasonic emitter, an ultrasoundtransducer, a piezoelectric ultrasound transducer, a compositetransducer, a capacitive micromachined ultrasound transducer, orcombinations thereof. In an aspect of the present invention, more thanone component for generating ultrasound waves is used and one or morecomponents may be found in an array configuration. In one aspect of thedisclosed methods, the component for generating ultrasound waves may bephysically attached to, wearably attached to, or implanted in thesubject. In an aspect of the present invention, the number of elementsof a component, such as the number of elements that comprise anultrasonic transducer or CMUT, that may be used in an ultrasound devicemay range from about 1 to 299 transducer or CMUT elements, from about 1to 1000 transducer or CMUT elements.

Methods for modulating cellular activity may be used with other methodsfor imaging or acting on a subject. For example, electroencephalogram,magnetoencephalography, magnetic resonance imaging, positron emissiontomography, computed tomography, or a combination may used withultrasound treatments to modulate cellular activity. Methods formodulating cellular activity may comprise a closed- or open-feedbackloop, and analyzing means, such as an algorithm or logic device, toevaluate feedback data from the subject and modify the ultrasoundstimulus waveform based on that feedback data. Methods may comprise useof a light emitting device. Methods for modulating cellular activity maybe repeated, such as performing the method of providing an ultrasoundstimulus waveform two or more times.

Methods for targeted cellular modulation comprises combined steps ofimaging a portion of the subject and then providing one or moreultrasound stimulus waveforms to modulate cellular activity, andparticularly cellular activity in the portion of the subject for whichimaging data had been obtained. For example, a method comprisesproducing ultrasound imaging data of a portion of a subject, wherein theultrasound imaging data was obtained by acoustically coupling at leastone component for generating ultrasound waves to an external surface ofa subject, driving at least one component for generating ultrasoundwaves to form at least one ultrasound waveform, wherein the waveformcomprises one or more frequencies, with an intensity in a range fromabout 0.0001 to about 900 mW/cm² and a frequency in a range from about0.01 to 5 MHz, at the site of the cells to be imaged, and using theultrasound data generated as a guide in determining where to provide atleast one ultrasound stimulus waveform, which comprises acousticallycoupling at least one component for generating ultrasound waves to anexternal surface of a subject, and driving at least one component forgenerating ultrasound waves to form at least one stimulus waveform,wherein the stimulus waveform comprises one or more frequencies, with anintensity in a range from about 0.0001 to about 900 mW/cm² and afrequency in a range from about 0.02 to about 1.0 MHz, at the site ofthe cells to be modulated. In an aspect of the invention, the ultrasoundwaves may be low-intensity ultrasound waves. Methods for modulatingcellular activity in targeted neuromodulation comprise using imagingdata to map information that may be used in ultrasound treatment formodulation of cellular activity in the brain. For example, neuronal orcellular metabolic activity may be mapped, or the time of a neurosignalmay be monitored in response to an ultrasound transmission.

Methods for modulating cellular activity by providing ultrasoundtreatment include modulating neuronal cellular activity and treating asubject with a brain tumor. In treating a subject with a brain tumor,the at least one component for generating ultrasound waves may comprise1 to 1000 ultrasound transducer elements. In treating a subject with abrain tumor, the temperature of the tissue in the brain tumor may remainbetween 30° C. and 44° C. during application of ultrasound, and may notexceed 40° C. for more than 10 seconds. The ultrasound treatment may beprovided to the subject in combination with another treatment includingbut not limited to surgery, chemotherapy, recombinant proteins, smallorganic molecules, or pharmaceutical agents.

The present invention discloses methods for modulating cellular activityin reducing consequences of secondary injury following a traumatic braininjury in a subject and the treatment may be repeated, may be given atany time beginning immediately after the traumatic brain injury, and maybe given to the subject in combination with another treatment fortraumatic brain injury. The present invention discloses methods formodulating cellular activity in treating a subject with a spinal cordinjury, treating a subject with migraine, treating a subject with backpain, treating a subject with Parkinson's disease, essential tremor,Alzheimer's disease, obsessive compulsive disorder, schizophrenia,bipolar disorder, depression or other disease or condition originatingin the central nervous system of a subject, treating a subject in aminimally conscious state, a coma, or a vegetative state, treating asubject with locked in syndrome, treating a subject with a cardiacarrhythmia or treating a subject with diabetes comprising providingultrasound treatment comprising acoustically coupling at least onecomponent for generating ultrasound waves to the subject, and driving atleast one component for generating ultrasound waves to form at least thestimulus waveform, wherein the stimulus waveform comprises one or morefrequencies, with an intensity in a range from about 0.0001 to about 900mW/cm² at the site of the cells to be modulated. In an aspect, theacoustic frequency may be delivered to the subject in a single orcombination of acoustic frequencies ranging from 0.05 to 50 MHz. Thestimulus waveform may be administered in a focused manner or in anunfocused manner. In the disclosed methods, the stimulus waveform may beadministered in a continuous wave or pulsed manner. In treatingdiabetes, the stimulus waveform may be provided to the pancreas or vagusnerve of the subject.

The present invention comprises ultrasound wave generating devices forlow intensity ultrasound transmission, wherein the ultrasound transducerforms a stimulus waveform with an intensity in a range of 0.001 to 900mW/cm² generated at the site of the tissue. The present inventioncomprises ultrasound stimulus waveforms for modulating cellular activitycomprising an intensity in a range from about 0.0001 to about 900 mW/cm²at the site of the cells to be modulated, one or more ultrasoundfrequencies in a range from about 0.02 to about 1.0 MHz, at the site ofthe cells to be modulated, and a plurality of single pulses, wherein asingle pulse has a pulse duration ranging from about 0.001 msec to about10 minutes, wherein single pulses are repeated at a pulse repetitionfrequency ranging from about 0.001 to about 10 KHz to producespatial-peak temporal-average intensities ranging from about 21 to about900 mW/cm² at the site of cells to be modulated, wherein single pulsecomprises between about 10 and about 50000 acoustic cycles, wherein theduration of the ultrasound transmission ranges from about 0.01 msec toabout 10 sec.

DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

The term “treating” refers to inhibiting, preventing, curing, reversing,attenuating, alleviating, minimizing, suppressing or halting thedeleterious effects of a disease and/or causing the reduction,remission, or regression of a disease. Those of skill in the art willunderstand that various methodologies and assays can be used to assessthe development of a disease, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression ofthe disease.

“Increase” is defined throughout as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,150, 200, 250, 300, 400, or 500 times increase as compared with basallevels or a control.

Example 1: Quantification of Synaptic Activity in Hippocampal SliceCultures

Hippocampal slice cultures were prepared from thy-1-synaptopHluorin(spH) mice. SynaptopHlourin is expressed in both excitatory andinhibitory hippocampal neurons of thy-1-spH mice. Synaptic vesiclerelease (exoytosis) at an individual release site from these mice isindicated by spH which fluoresces at a particular wavelength (about 530nm) in the green portion of the optical spectrum when excited by laserlight at 488 nm from laser-scanning confocal microscope. The intensityof fluorescent emissions (F) from all sites in view is measured toquantify synaptic activity. ΔF expressed as a percentage indicatespercentage changes in total intensity of the fluorescence; and thereforepercentage changes in synaptic activity. Synaptic activity is indicatedby spH in both a CA1 stratum radiatum (CA1 SR) region and a CA1 stratumpyramidale (CA1 SP) region—the latter a region with a particularly highdensity of inhibitory synapses. Thus modulation of neural activityindicated by spH can indicate excitatory or inhibitory modulation orsome combination

Example 2: Comparison of Effects of Neural Activity Inducement BetweenUltrasound and Conventional Means

To determine whether the effects of ultrasound modulation are comparableto neural activity changes induced by more invasive, conventional means,consider FIG. 6A and FIG. 6B. FIG. 6A is a graph 600 that illustratescomparative temporal responses of neural activity after modulation byelectrical impulses and after modulation by an ultrasound waveform,according to an embodiment. The horizontal axis 602 is time in seconds;and the horizontal scale is given by segment 601 that corresponds to 5seconds. The vertical axis 604 indicates ΔF in percent (%); and thevertical scale is given by segment 605 that corresponds to 10%. Thestart of USW-1 is indicated by tick 603. Curve 610 indicates the averagetemporal response from USW-1, from over 148 individual responses, asgiven by average response 540 in graph 530. Curve 620 a indicates theresponse of spH fluorescence in Schaffer collaterals regions of neuraltissue to electrical stimulation using monopolar electrodes at 40 actionpotentials (AP) and 20 Hz (averaged over n=51 individual synapses).Similarly, curves 620 b and 620 c indicate the response of spHfluorescence in Schaffer collaterals to electrical stimulation usingmonopolar electrodes at 100 AP/20 Hz (n=63) and 250 AP/50 Hz (n=48),respectively.

Thus, graph 600 shows that the time rate of change (kinetics) andamplitudes of ultrasound triggered spH response, indicated by curve 610,are on the same order as those obtained in response to electricalstimulation, curves 620. The response is also similar to spH responsespreviously reported for different stimuli. This indicates thatultrasound is as effective as electrical stimulation for the treatmentof neural disease and disorders.

FIG. 6B is a graph 650 that illustrates temporal electrical responses ofa neuron after modulation by an ultrasound waveform, according to anembodiment. The horizontal axis 652 is time in seconds; and thehorizontal scale is given by segment 651 that corresponds to 1 second.The vertical axis 654 indicates voltage difference across the neuronalmembrane in milliVolts (mV); and the vertical scale is given by segment655 that corresponds to 50 mV. Here a different ultrasound waveform,referenced as USW-2 is used. USW-2 consists of five pulses, each at 0.44MHz for 10 square wave cycles for a pulse length of 22.7 μs. The PRF isa constant 10 Hz, so the entire waveform lasts just over 0.5 seconds(one tenth the duration of USW-1). The start of USW-2 is indicated bytick 653.

Trace 660 indicates action potentials (e.g., membrane voltage) inresponse to pulsed ultrasound waveforms during whole-cell current clamprecordings of CA1 pyramidal neurons. The trace 660 includes five spikes662 that represent neural firing (transmission of an electrical pulse)along the neuron during the 0.5 seconds of the waveform. However, ingeneral, whole-cell electrophysiological approaches were not very usefulin studying ultrasonic neuromodulation due to electrode resonationsproducing loss of whole-cell seals during ultrasound waveformpropagation. Thus, graph 650 shows that the time rate of change(kinetics) and amplitudes of ultrasound membrane potential indicated bytrace 660 shows neuron firing induced by low intensity ultrasoundwaveforms.

Cavitation or other evidence of gross membrane damage was not observedat the low intensities used herein. Slice cultures prepared fromthy-1-YFP mice 10 were chronically modulated with USW-1 every 8 minutesfor 36-48 hours. The membrane structures of YFP+ neurons receiving suchchronic ultrasound modulation were similar to unmodulated controls.According to an embodiment, chronic modulation with ultrasound waveformsdoes not cause gross membrane damage in CA1 SP region. Usinghistological techniques, no indications of cell damage are evident.Histology indicates the presence of fine structures such as dendriticspines for both control and chronically modulated conditions.Chronically stimulated neurons appear to have more dendrites. Thus, insome embodiments, ultrasound waveforms are repeated for sufficientduration to modulate neuronal morphology in subtle ways which canmediate neural function.

Thus, low intensity pulsed ultrasound waveforms appear to be highlyeffective in modulating neural activity, safe for prolonged use, andable to stimulate desirable changes in neuronal morphology.

Example 3: Stimulation of SNARE-Mediated Synaptic Vesicle Exocytosis andSynaptic Transmission by Low-Intensity, Low-Frequency Ultrasound

Changes in membrane tension produced by the absorbance of mechanicalenergy (e.g., sound waves) alter the activity of individual neurons dueto the elastic nature of their lipid bilayers and spring-like mechanicsof their transmembrane protein channels. In fact, many voltage-gated ionchannels, as well as neurotransmitter receptors possess mechanosensitiveproperties permitting them to be differentially gated by changes inmembrane tension. Therefore, a set of methods for investigating theinfluence of mechanical energy conferred on neuronal activity byultrasound were utilized. Using these methods, it was found that pulsedultrasound is capable of stimulating SNARE-mediated synaptictransmission, as well as voltage-gated sodium (Na⁺) and (calcium) Ca²⁺channels in central neurons. SNARE proteins are a class of proteins thatinclude neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx1A), and Synaptotagmin I (Syt I) that play a role in synaptictransmission and vesicle exocytosis. These measurements utilized thefollowing procedures.

Hippocampal slice cultures were taken from postnatal day 7-8 thy-1-spH,thy-1-YFP, or wild-type mice in a manner similar to previously describedmethods. Briefly, transverse hippocampal slices (about 400 μm thick)were prepared with a wire slicer (MX-TS, Siskiyou, Inc., Grants Pass,Oreg., USA) and maintained in vitro on Millicell-CM filter inserts(PICMORG50, Millipore, Bedford, Mass.) in a 36° C., 5% CO₂, humidified(99%) incubator. Slices were used for experiments between 7 and 12 daysin vitro. To cleave SNARE-proteins in some experiments, BoNT/A at 250nanograms per milliliter (ng/mL, 1 ng=10⁻⁹ grams and 1 mL=10⁻³ Liters)was added to the slice culture media 24-36 hours prior to experiments.

Following CO₂ inhalation, mice were rapidly decapitated and their entirebrains were removed, the dura was carefully removed, and the brain wasthen placed in ice-cold dissection artificial CSF (aCSF) containing 83milliMoles (mM, 1 mM=10⁻³ Moles of a compound) NaCl, 2.5 mM KCl, 3.3 mMMgSO₄, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃, 22 mM glucose, 72 mM sucrose, and0.5 mM CaCl₂, and equilibrated with 95% O₂/5% CO₂. Brains were allowedto recover for 5 minutes in ice-cold aCSF before recovering for about 20minutes at 37° C. before being bulk loaded for some experiments withOGB-1 AM dye at room temperature (21-23° C.).

In order to load slice cultures prepared from wild-type mice with CoroNaGreen AM (Invitrogen, Carlsbad, Calif., USA) 5 microLiters (AL, 1μL=10⁻⁶ Liters) 20% Pluronic F-127 in DMSO (Invitrogen) was added to a50 microgram (μg, 1 μg=10⁻⁶ grams) vial of CoroNa Green AM. The dyesolution was then vortexed for 15 minutes before adding 100 μL culturemedium. Then 5 μL of the dye-containing solution was added to 1 μLculture medium underneath culture inserts, as well as adding 5 μL to thesurface of slices. Following a 10 minute loading time at 36° C., sliceswere washed three times with slice culture medium, allowed to recover anadditional 10 minutes, and then used for experiments. To load slicecultures with OGB-1 AM (Invitrogen), 2 μL, 20% Pluronic F-127 in DMSO(Invitrogen) and 8 μL was added to a 50 μg vial of OGB-1 AM. Thedye-containing solution was then vortexed for 30 minutes before adding90 μL culture medium. Then 20 μL of this dye-containing solution wasadded to 3 mL culture medium and slices were incubated in this solutionfor 30-40 minutes at 37° C. Slices were washed three times with sliceculture medium, then loaded with sulforhodamine 101 (Invitrogen; 10 μMin slice culture medium for 15 minutes) or allowed to recover for 30minutes before being used in experiments. In order to load ex vivobrains with OGB-1 AM we used a procedure similar to that describedabove, but substituted the slice culture medium for dissection aCSF (seeabove)—60 μl of the dye-containing solution was added to 9 mL dissectionaCSF. Brains were loaded for 30 minutes at room temperature then rinsedthree times and allowed to recover for an additional 30 minutes at roomtemperature before use.

Slice cultures or whole ex vivo brains were transferred to a recordingchamber containing normal aCSF. Normal aCSF contains 136 mM NaCl, 2.5 mMKCl, 1.3 mM MgSO₄, 10 mM HEPES, 10 mM glucose, and 2.5 mM CaCl₂), pH 7.4at room temperature. Recording chambers were affixed over transducers ona custom built-stage on an Olympus Fluoview FV-300 laser-scanningconfocal microscope (Olympus America, Inc., Center Valley, Pa., USA).Excitation of spH, OGB-1 AM, and CoroNa Green AM was performed using a488 nanometer (nm, 1 nm=10⁻⁹ meters) optical wavelength of an argonlaser and in some experiments DiI was excited using a 546 nm HeNe laser.Time-series images were acquired using 20× (lens with 0.5 numericalaperture, NA) or 40× (0.8 NA) Olympus UMPlanFL water-immersion lenses.

Slice recording chambers consisted of culture inserts and a constructedaCSF reservoir held in place either by vacuum grease or superficialtension between the silicon face of transducers and the insert. Thisapproach produced a 4.5 mm standoff distance between the face of thetransducer and the imaging plane on the surface of slices. In somecases, to test remote transmission of ultrasound waveforms on neuronalactivity, slice cultures were mounted at the top of an aCSF column in a500 mL beaker containing immersed transducers, which were affixed to thebottom beakers providing a 45 mm standoff distance. The ventral (bottom)surface of whole ex vivo brains were glued to the bottom of polystyrene6-well plates using superglue, which were filled with aCSF and mountedon transducers using ultrasonic coupling gel. Confocal imaging of OGB-1in ex vivo brains was conducted on the superficial dorsal (top) surfaceof ex vivo brains during and after transmission of pulsed ultrasoundwaveforms through the brain from the ventral surface.

Whole-cell current clamp recordings from visually identified CA1pyramidal neurons were performed using standard approaches. Briefly,patch electrode pipettes filled with an intracellular solutioncontaining 130 mM KCl, 10 mM Na-HEPES, 10 mM Di-Tris-P-creatine, 0.2 mMEGTA, 3 mM Mg-ATP, and 0.5 mM Na-GTP, 280-290 mM mOsm, pH 7.2; the finalresistance of these unpolished patch electrodes was 5-7 megaOhms (MΩ, 1MΩ=10⁶ ohms). Current clamp recordings were performed using a MultiClamp700B patch-clamp amplifier with pCLAMP 10 software (Molecular Devices,Sunnyvale, Calif., USA). Following 5-10 minutes of whole-cell access,changes in membrane voltage were recorded in response to stimulationwith pulsed ultrasound waveforms.

Confocal images were analyzed offline using ImageJ (see, e.g.,http://rsb.info.nih.gov/ij/) or the Olympus Fluoview 5.0 software. Weexpress changes in spH fluorescence as a percent change from baselinefluorescence levels. For OGB-1 and CoroNa Green signals, ΔF/F₀ wascalculated using standard approaches ΔF=F−F₀. Characteristics ofultrasound waveforms and electrophysiological analyses were performedoffline using Igor Pro (WaveMetrics, Lake Oswego, Oreg., USA). Datashown are mean±S.E.M. The resulting measurements provide insight intothe mechanisms for ultrasound control on neural activity.

Transmission of USW-1 into slices triggered synaptic vesicle exocytosisproducing a AF due to spH of 18.52%±2.2% at individual release sites(148 samples) in CA1 stratum radiatum (as shown above in FIG. 5 ).Several other pulsed ultrasound waveforms were identified, which wereeffective at triggering synaptic vesicle release as listed below inTable 1. For example, an ultrasound waveform composed of pulses withf=0.67 MHz, PL=74.5 μs, c/p=50,000 delivered at PRF=10 Hz with Np=5 (fora 0.5 duration) also stimulated synaptic vesicle release, as indicatedby an AF due to spH=12.86%±2.6%, for 74 samples.

TABLE 1 Effectiveness of Low Intensity Pulsed Waveforms in StimulatingSynaptic Vesicle Release According to Multiple Embodiments p-p sq. Fwave spH re- PL c/p (Mhz) PRF Np amplitude sponse 22.7 μs 10 0.44 5 sramp 250 500 mV + 0-100 Hz 74.5 ms 50,000 0.67 10 Hz 5 150 mV + 74.5 ms50,000 0.67 10 Hz 150 100 mV + 22.7 μs 10 0.44 5 s ramp 250 100 mV −0-100 Hz 2.27 μs 1 0.44 5 s ramp 250 500 mV − 0-100 Hz 11.35 μs 5 0.44 5s ramp 250 500 mV + 0-100 Hz 11.35 μs 5 0.44 5 s ramp 250 100 mV − 0-100Hz 2.27 μs 1 0.44 20 Hz 100 500 mV − 22.7 μs 10 0.44 20 Hz 100 500 mV +22.7 μs 10 0.44 250 Hz  250 500 mV − 22.7 μs 10 0.44 10 s ramp 500 500mV + 0-100 Hz 22.7 μs 10 0.44 15 s ramp 750 500 mV + 0-100 Hz 113.5 μs50 0.44 5 s ramp 25 500 mV − 0-10 Hz 113.5 μs 50 0.44 5 s ramp 500 500mV + 0-200 Hz 113.5 μs 50 0.44 5 s ramp 250 500 mV + 0-100 Hz 170.25 μs75 0.44 5 s ramp 250 500 mV + 0-100 Hz 227.0 μs 100 0.44 5 s ramp 250500 mV + 0-100 Hz 22.7 μs 10 0.44 50 Hz 250 500 mV −

Using a DiIoistic labeling approach to visualize dendritic spines,populations of putative excitatory terminals were examined. Nodifference was found between the ultrasound induced ΔF due to spHobtained from terminals impinging on dendritic spines and somaticsynapses impinging on cell bodies. The spine synapses showed ΔF due tospH=19.94%±1.7%; and somatic synapses ΔF due to spH=20.55%±2.7%, with 45samples for each.

To determine the mechanisms affected by low intensity ultrasoundwaveforms, various inhibitors known to affect certain processes wereintroduced to the slices of neural tissue. For example, SNARE-mediatedexocytosis is inhibited by BoNT/A. Introducing 250 ng/mL BoNT/A nearlyabolished exocytosis in response to low intensity ultrasound waveforms.Thus it is concluded that low intensity ultrasound waveforms exciteSNARE-mediated exocytosis. Sodium ion (Na⁺) conductance is inhibited byvoltage gated Na⁺ channel pore blocker tetrodotoxin (TTX). Introducing 1μM of TTX nearly abolishes exocytosis in response to low intensityultrasound waveforms. Thus it is concluded that low intensity ultrasoundwaveforms relies on Na conductance. Synaptic transmission other thanexocytosis is blocked by CNQX and APV. Adding 20 μM CNQX and 100 μM APVblocked excitatory network activity and reduced ΔF due to spH by about 6percentage points (50% of the low intensity ultrasound effect), thusindicating that low intensity ultrasound waveforms stimulates synaptictransmission and not just exocytosis.

FIG. 7 is a graph 700 that illustrates example effects of some processinhibitors on neural activity modulated by an ultrasound waveform,according to an embodiment. The horizontal axis 702 is time in seconds;and the horizontal scale is given by segment 701 that corresponds to 5seconds. The vertical axis 704 indicates ΔF in percent (%); and thevertical scale is given by segment 705 that corresponds to 5%. The startof USW-1 is indicated by tick 703. Curve 710 indicates the averagetemporal response to USW-1 when no process inhibitors are introduced tothe neural tissue. Curve 730 indicates the average temporal responsewhen 20 μM CNQX and 100 μM APV are added to block excitatory networkactivity, reducing the effect of USW-1 by half to about 6%. Curve 740indicates the average temporal response when 1 μM of TTX is added toinhibit sodium (Na⁺) conductance, nearly abolishing the effect of USW-1.Curve 750 indicates the average temporal response when 250 ng/mL BoNT/Ais added to inhibit SNARE-mediated exocytosis, again nearly abolishingthe effect of USW-1.

Example 4: Stimulation of Voltage-Dependent Calcium Transients inNeurons by Low-Intensity, Low-Frequency Ultrasound

Using the Na⁺ indicator CoroNa Green AM, as known in the art, incultures prepared from wild-type mice, it was found that USW-1 triggeredNa⁺ transients in CA1 pyramidal neurons (CA1 SP). According to anembodiment, FIG. 8 is a graph 810 that illustrates an example temporaleffect on neural Na transients after modulation by an ultrasoundwaveform, according to an embodiment. The horizontal axis 812 is time inseconds; and the horizontal scale is given by segment 811 thatcorresponds to 5 seconds. The vertical axis 814 indicates ΔF due toCoroNa Green in percent (%); and the vertical scale is given by segment815 that corresponds to 4%. The start of USW-1 is indicated by tick 813.Individual responses of Na transients after each ultrasound waveform aregiven by traces 820 and the average response indicated by curve 822. Themaximum response was ΔF/F₀=5%±0.6% for n=18 measurements. This responsewas blocked by the addition of tetrodotoxin (TTX) as indicated by theindividual responses 840.

To determine if pulsed ultrasound waveforms were also capable ofactivating Ca²⁺ transients, slice cultures prepared from wild-type micewere loaded with the Ca2⁺-indicator Oregon Green 488 BAPTA-1 AM (OGB-1AM) and Sulforhodamine 101 to differentiate between neurons and glialcells, as known in the art. According to an embodiment, USW-1 activationof Ca2⁺ transients in both neurons and glial cells can be visualizedusing histological techniques that include, but are not limited to, theuse of green fluorescence from OGB-1 in neurons, and by yellowfluorescence from Sulforhodamine in glial cells.

FIG. 9 is a graph 920 that illustrates example temporal effects onneural and glial Ca transients after modulation by an ultrasoundwaveform, according to an embodiment. The horizontal axis 922 is time inseconds; and the horizontal scale is given by segment 921 thatcorresponds to 20 seconds. The vertical axis 924 indicates ΔF in percent(%); and the vertical scale is given by segment 925 that corresponds to100% for neurons and segment 926 that corresponds to 120% for glialcells. The zero value is offset for each set of curves to visuallyseparate them. The start of USW-1 is indicated by tick 923. Individualresponses of Ca²⁺ transients after each of four USW-1 instances aregiven by traces 950 a, 950 b, 950 c and 950 d for neurons and by traces960 a, 960 b, 960 c and 960 d for glial cells, respectively. Forneurons, F/F₀=114%±10% in 61 samples. For glial cells, ΔF/F₀=140%±12%for 55 samples. There are some differences in the time rate of changebetween the two cell types.

Modulation with USW-1 also induced presynaptic Ca²⁺ transients in CA1SR. According to an embodiment, USW-1 activation of presynaptic Ca²⁺transients in CA1 SR can be visualized using histological techniquesthat include, but are not limited to, the use of green fluorescence fromOGB-1 in neurons. The observed ΔF/F₀=76%±7% for 31 samples.

Example 5: Activation of Voltage-Gated Sodium Channels in Neurons byLow-Intensity, Low-Frequency Ultrasound

To determine whether ultrasound triggered Ca²⁺ transients are primarilymediated by voltage-gated Ca²⁺ channels, Cd⁺⁺ was added to block voltagegated Ca²⁺ channels. Adding 500 μM Cd⁺⁺ nearly abolished OGB-1 signalsin response to USW-1. Likewise, the addition of TTX blocked about 85% ofthe OGB-1 signal produced by USW-1. FIG. 10 is a graph 1030 thatillustrates an example temporal effect on neural presynaptic activity bymodulation with an ultrasound waveform, according to an embodiment. Thehorizontal axis 1032 is time in seconds; and the horizontal scale isgiven by segment 1031 that corresponds to 5 seconds. The vertical axis1034 indicates ΔF of OGB-1 in percent (%); and the vertical scale isgiven by segment 1035 that corresponds to 70%. The start of USW-1 isindicated by tick 1033. Curve 1040 indicates the average presynaptictemporal response of OGB-1 to USW-1 when no process inhibitors areintroduced to the neural tissue. Curve 1050 indicates the averagetemporal response when TTX is added to inhibit Na⁺ conductance, nearlyabolishing the effect of USW-1. Curve 1060 indicates the averagetemporal response when Cd⁺⁺ is added to block voltage-gated Ca²⁺channels, again nearly abolishing the effect of USW-1. Residual Ca²⁺transients not blocked by Cd⁺⁺ or tetrodotoxin (TTX) are likely toinvolve other Ca²⁺ sources such a NMDA or TRPC 1 receptors, whichinterestingly both possess mechanosensitive properties and are expressedin hippocampal neurons. Using ultrasound waveforms with shorter duration(e.g., f=0.44 MHz, PL=0.18 ms, c/p=80, PRF=10 Hz, and Np=3), Ca²⁺transients were observed in neurons (ΔF/F₀=38%±2%, for 24 samples) withfaster kinetics.

Since salt-containing solutions confer low acoustic impedance (−1.56×10⁶N·s/m³), Ca²⁺ transients were observed in response to low intensitypulsed ultrasound waveforms even when transducers were placed 45 mm awayfrom slices (data not shown). Soft biological tissues (including brain)have acoustic impedances ranging from 1.5-1.8×10⁶ N·s/m³. To determinewhether Ca²⁺ responses could be obtained by transmitting low intensitypulsed ultrasound through intact brain, OGB-1 fluorescence was measuredon the dorsal surface of ex vivo brains obtained from wild-type adultmice while transmitting low intensity pulsed ultrasound waveformsthrough their ventral surfaces. In this ex vivo brain preparation, Ca²⁺transients were observed in response to low intensity pulsed ultrasoundwaveforms, which were similar to those observed in slice cultures.

Enhanced effects were noted for some low intensity waveforms. Forexample, one such low intensity waveform included three pulses (Np=3) atPRF=10 Hz with c/p=80, but with different ultrasound frequencies atalternating pulses, e.g., f=0.44 MHz in the first and third pulse andf=0.67 MHz in the second pulse. This pulse produced substantiallygreater excitation of neural activity compared to other waveforms testedat an intensity less than 10 mW/cm².

Example 6: Examination of the Influence of Ultrasound Stimulus Waveformson Intact Brain

To conduct transcranial ultrasound (US) stimulation of intact motorcortex, mice were anesthetized using a ketamine-xylazine cocktail (70mg/kg ketamine, 7 mg/kg xylazine) administered intraperitoneally. Thehair on the dorsal surface of the head over regions corresponding tomotor cortex was trimmed using microdissection scissors. Mice were thenplaced in a Cunningham mouse stereotax and affixed to a vibrationisolation table. Ultrasound transducers with affixed focusing guideswere lowered to points above the skin corresponding to motor cortexidentified using standard coordinates. Focusing tubes were then placedon the dorsal surface of the skin above motor cortex and acousticallycoupled to the skin using ultrasound coupling gel. Transcranial pulsedUS stimulus waveforms were delivered to the targeted motor cortex usinga standard TTL triggering protocol and a digital I/0 device (Digidata1440; Molecular Devices, Sunnyvale, Calif., USA) connected to a PCcontrolled using pClamp software (Molecular Devices) to activatefunction generators. TTL signal markers indicated the onset and lengthof US stimulus waveforms. Video recordings of stimulation trials wereacquired using a standard webcam. During stimulation trials,electrophysiological data (multi-unit activity (MUA), LFP, and EMG wereacquired; see below). Following stimulation, animals were either allowedto recover from anesthesia or processed as described in the materialsand methods below.

More specifically, low-intensity US waveforms were transmitted throughacoustic focusing guides to the intact motor cortex of anesthetized mice(n=127). FIG. 18A shows an illustration of the method used to transmitlaterally focused US stimulus waveforms to intact mouse motor cortex.The optimal gains between transcranial transmission and brain absorptionoccurs for US at acoustic frequencies (f)<1.0 MHz. Thus, transcranialstimulus waveforms in the frequency range of 0.25 to 0.50 MHz wereconstructed, while also varying intensity and using 80 to 225 cyclepulses.

FIGS. 18B and 21 show examples of the strategy and parameters used inconstructing low-intensity US stimulus waveforms. Intensities generatedby the illustrated stimulus waveform are given in the yellow-box.Ultrasound (US) stimulus waveforms were constructed using methodssimilar to those previously described. Twenty-five (25) mm diameter,water-matched, broadband US transducers having a center frequency of 0.5MHz (V301-SU, Olympus NDT, Waltham, Mass., USA) were used. Ultrasound(US) pulses were generated by brief bursts of square waves (0.2 μsec;0.5 mV peak-to-peak) using an Agilent 33220A function generator (AgilentTechnologies, Inc., Santa Clara, Calif., USA). Square waves were furtheramplified (50 dB gain) using an ENI 240L RF amplifier. US pulses wererepeated at a pulse repetition frequency by triggering the abovereferenced function generator with a second Agilent 33220A functiongenerator.

Single ultrasound pulses had pulse durations (PD) lasting from 0.16 to0.57 msec, peak rarefactional pressures (p_(r)) of 0.070 to 0.097 MPa,pulse intensity integrals (PII) of 0.017 to 0.095 mJ/cm², andspatial-peak pulse-average intensities (I_(SPPA)) of 0.075 to 0.229W/cm². Single US Pulses were repeated at pulse repetition frequencies(PRF) ranging from 1.2 to 3.0 KHz to produce spatial-peaktemporal-average intensities (I_(SPTA)) of 21 to 163 mW/cm² fortranscranial stimulus durations of 26 to 333 msec. The above reportedintensities were measured transcranially at points corresponding tointact motor cortex in fresh ex vivo heads. It was observed that <10%intensity loss due to transmission of US waveforms through the hair,skin, skull, and dura of mice (FIG. 22 ).

In FIG. 22 , a single cycle (top) and 100 cycle pulse (bottom) of 0.5MHz was transmitted through ultrasonic coupling gel from the transducerface directly to the face of a hydrophone (left) or through a fresh exvivo head (right) containing hair, skin, skull, and dura to the face ofa hydrophone.

Table 2 provides an overview of the US stimulus waveforms used in thisExample. In Table 2, a single asterisk (*) indicates that standard USwaveforms were used during the investigation. In Table 2, a doubleasterisk (**) indicates relatively high-intensity US waveforms were usedto assess safety.

TABLE 2 Low-Intensity Transcranial Ultrasound (US) Waveform Propertiesused to Stimulate Intact Mouse Motor Cortex PD PRF US Stim P_(r) P_(II)I_(SPPA) I_(SPTA) f c/p (msec) (KHz) Np length (sec) (MPa) (mJ/cm²)(W/cm²) (mW/cm²) MI *0.250 120 0.480 1500 80 0.053 0.089 0.082 0.167123.40 0.18 0.250 80 0.320 1200 100 0.083 0.088 0.051 0.161 60.78 0.180.350 200 0.571 1500 40 0.027 0.071 0.044 0.077 65.98 0.12 0.350 1800.514 1500 80 0.053 0.072 0.040 0.077 59.38 0.12 *0.350 80 0.229 2500150 0.060 0.070 0.017 0.075 42.90 0.12 0.350 120 0.343 1500 80 0.0530.071 0.027 0.076 40.76 0.12 0.350 125 0.357 1200 100 0.083 0.071 0.0270.076 32.61 0.12 0.350 100 0.286 1500 80 0.053 0.072 0.022 0.076 32.400.12 0.350 80 0.229 1200 100 0.083 0.070 0.017 0.075 20.59 0.12 0.425100 0.235 3000 1000 0.333 0.082 0.025 0.107 74.97 0.13 0.425 80 0.1883000 150 0.050 0.083 0.019 0.103 57.80 0.13 0.425 80 0.188 3000 8000.267 0.083 0.019 0.103 57.80 0.13 0.425 80 0.188 2750 200 0.073 0.0830.019 0.103 52.98 0.13 0.425 80 0.188 1500 200 0.133 0.083 0.019 0.10328.90 0.13 0.500 150 0.300 2500 150 0.060 0.097 0.065 0.225 162.74 0.14**0.500 225 0.450 1500 80 0.053 0.092 0.095 0.216 142.20 0.13 0.500 2000.400 1500 40 0.027 0.095 0.087 0.223 130.17 0.13 0.500 125 0.250 1500100 0.067 0.097 0.054 0.225 80.78 0.14 *0.500 100 0.200 1500 80 0.0530.097 0.043 0.228 64.53 0.14 0.500 80 0.160 1200 100 0.083 0.097 0.0340.229 40.81 0.14

The influence of US stimulus waveforms was examined on intact brainactivity by recording multi-unit activity (MUA) from the primary motorcortex (M1) of mice (n=6). Low-intensity US stimulus waveforms focallyrestricted to unilateral M1 induced a significant increase in thefrequency of cortical spiking in a temporally precise manner (ANOVA,F_(19, 480)=69.72, P<0.001; FIG. 18C and FIG. 23 ). Delivery of USstimulus waveforms to M1 produced local field potentials (LFP) with meanamplitudes of −350.59±43.34 μV (FIG. 18C). Application of tetrodotoxin(TTX) to the motor cortex blocked US-evoked cortical activity,indicating that transcranial US elicits action potentials mediated byvoltage-gated sodium channels (FIG. 18C; raw control (black), averagecontrol (blue), and average TTX (red)).

Example 7: Determination of the Area of Intact Motor Cortex Activated byUltrasound

Using a c-fos labeling technique, studies were conducted in order todetermine the area of motor cortex activated by laterally focused USstimulus waveforms (n=5 mice). An ANOVA revealed a significantly higherdensity of activated cells in the targeted motor cortex compared toadjacent untargeted motor and primary somatosensory (S1) cortex(F_(4,45)=17.1, P<0.001; FIG. 24A). Further densiometric analysesrevealed significantly larger clusters of activated cells in targeted M1compared to the untargeted and adjacent S1 (targeted M1 clusterarea=9.45±1.81 mm², untargeted S1 cluster area=3.01±1.54 mm²; T-test,P<0.05; FIG. 24B). The size of the activated area is consistent with theimplemented US wavelengths (≈3-6 mm in brain tissue), as well as thearea of acoustic pressure fields produced by the laterally restrictedspatial envelope of US stimulus waveforms generated by focusing guides(≈8-10 mm²; FIG. 25 ).

Multi-dimensional pressure output profiles obtained using a 25 mm planarUS transducer alone (top) and with an attached 5 mm diameter focusingguide (middle) or an attached 5 mm guide tapered to a 2 mm outputdiameter (bottom) used to differentially target US stimulus waveforms tothe motor cortex were obtained. FIG. 25 shows the normalized pressureprofiles obtained with a 25 mm planar transducer (black) and with a 5 mm(blue) or tapered 2 mm output diameter (red) focusing guide attached areillustrated for X (top) and Y (bottom) planes. The full-widthhalf-maximum (FWHM) values are illustrated for each plane.

Example 8: Determination of Effects of Ultrasound on Intact CentralNervous System Activity

To determine the effects of low-intensity transcranial US on intactcentral nervous system activity (CNS) activity, its influence on knowndescending corticospinal motor circuits was investigated. Fine-wireelectromyogram (EMG) recordings of muscle activity in response to thetranscranial delivery of US stimulus waveforms to intact motor cortexwere acquired (n=43 mice; FIGS. 18D and 18E). For example, in FIG. 18D,the schematic (top) illustrates the experimental approach to stimulatingdescending corticospinal tracts with transcranial ultrasound. Rawelectromyogram (EMG) traces (bottom) in response to right transcranialM1 stimulation illustrate US-evoked activity of the left tricepsbrachii. FIG. 18E shows that raw (left) and full-wave rectified (FWR;right) EMG traces for a spontaneous (top) and average (10 trials)US-evoked (bottom) event. The duration of the US stimulus waveform(black), average US-evoked EMG trace (grey), and EMG integral (green)are superimposed at lower-right.

Descending corticospinal motor circuits were successfully stimulated in90.3% of the cases. Bilateral US stimulation of motor cortex producedbilateral motor activation of several muscle groups. Transcranial USdelivered through focusing guides, however, led to selective activationof muscle groups depending on the cortical region targeted. For example,targeted unilateral stimulation of right M1 triggered left forelimbmuscle contractions sufficient to induce paw movements (FIG. 18D). Inmost cases, the location of US focusing guides over motor cortex waschanged to produce differential movement behaviors (whisker movementscompared to forelimb and tail movements. Although the spatial resolutionfor focusing US is currently limited by the acoustic wavelengthemployed, recent advances in focusing US with adaptive optics permit USto gain spatial resolutions below the diffraction limits as has beenachieved in light microscopy.

Unilateral US stimulation of motor cortex triggered EMG activity in thecontralateral triceps brachii muscle (n=17 mice) with a mean responsewith latency of 20.88±1.46 msec and was consistent across trials. FIG.19A shows EMG response latency of left triceps brachii in response toright MI activation is plotted as a function of repetitive trial number(left) for a 10 sec ITI. Individual US-evoked raw EMG traces are shownfor different trials (right). Bilateral stimulation of motor cortex totrigger tail movements was similarly consistent and elicited EMGactivity in the lumbosacrocaudalis dorsalis lateralis muscle (n=26 mice)with a response latency of 22.65±1.70 msec. These response latencies areconsistent with the observations of others using optogenetic andelectrical methods to stimulate motor cortex. The repeatability of motoractivation functioned as the inter-trial interval (ITI) betweenUS-stimulus events decreased was studied next. An ANOVA revealed the EMGfailure probability significantly (F_(3, 92)=120.40; P<0.001) increasedas the ITI was reduced. FIG. 19B shows EMG failure probabilityhistograms are shown for four progressively decreasing ITIs (left). RawUS-evoked EMG traces are shown for two different ITI times (right). Todetermine if US-evoked cortical activity drives the EMG response, insome experiments tetrodotoxin (TTX) was applied to the motor cortexduring stimulation trials. It was observed that the application of TTXto motor cortex blocked EMG activity, which indicates transcranial USelicits cortical action potentials to stimulate motor circuit activityand peripheral muscle contractions (n=4 mice). FIG. 19C shows raw EMGtraces illustrating that application of TTX to the motor cortex blocksUS-evoked descending corticospinal circuit activity.

Example 9: Evaluation of the Effects of Ultrasound on Brain Temperature

To evaluate the influence of low-intensity pulsed ultrasound on braintemperature, however, the temperature of motor cortex was monitoredduring transcranial ultrasound transmission while varying acousticintensity and pulse duration (PD) times. Equations for estimatingthermal absorption of US in biological tissues predict that 0.5 MHz USpulses exerting a p_(r) of 0.097 MPa for a PD of 0.57 msec can induce atemperature increase of 2.8×10⁻⁶° C. in brain. Briefly, the maximumtemperature change (ΔT_(max)) is estimated to be:

${\Delta T_{{ma}\; x}} = \frac{Q\;\Delta\; t}{C_{V}}$where Δt is the pulse exposure time, where C_(v) is the specific heatcapacity for brain tissue≈3.6 J/g/K and where Q is the rate at whichheat is produced defined by (Nyborg 1981):

$Q = \frac{\alpha p\frac{2}{0}}{p0}$where p is the density of the medium, c is the speed of sound in themedium as described above, where α is the absorption coefficient ofbrain (≈0.03 Np/cm for 0.5 MHz ultrasound), and p₀ is the pressureamplitude of US stimulus waveforms.

In some experiments, prior to transmitting transcranial ultrasoundwaveforms into intact brains, a small craniotomy (d≈2 mm) was performedon mouse temporal bone. Following removal of dura, a 0.87 mm diameterthermocouple (TA-29, Warner Instruments, LLC, Hamden, Conn., USA) wasinserted into motor cortex through the cranial window. The thermocouplewas connected to a monitoring device (TC-324B, Warner Instruments),which was connected to the Digidata 1440A in order to record temperature(calibrated voltage signal=100 mV/° C.) using pClamp connected to a PC.To facilitate off-line analyses, TTL signal markers indicated the onsetof US stimulus waveforms.

All US stimulus waveforms used had p_(r) values <0.097 MPa and PD times≤0.57 msec. None of the US waveforms used to stimulate cortical activityelicited a significant change in cortical temperature within theresolution limits (FIG. 19D). Under these experimental conditions, USpulses with p_(r) values of 0.1 MPa and PD times >50 msec were requiredto produce a temperature change (ΔT) of≈0.02° C. FIG. 19D shows the raw(black) and average (grey) temperature recordings from M1 in response totransmission of US waveforms with different intensity characteristics.FIG. 19D illustrate stimulus waveforms (top) do not produce an increasein cortical temperature as observed with higher intensity waveforms(middle and bottom). These observations bolster the idea of apredominantly mechanical (nonthermal) mechanism of action and highlightthe safety margins of stimulus waveforms.

Example 10: Determination of Effects of Variations of the AcousticFrequency and Intensity of Ultrasound on Neuronal Circuit Activity

Experiments were designed in order to determine how the acousticfrequency and intensity of US stimulus waveforms influenced neuronalcircuit activity. The effect of four different US frequencies (0.25,0.35, 0.425, 0.500 MHz) on EMG amplitudes produced by triceps brachii inmice was examined (n=20). A two-way ANOVA revealed a significant maineffect of US frequency on EMG amplitudes (F_(3, 1085)=3.95, P<0.01)whereby lower frequencies produced more robust EMG responses (FIG. 19E).To better understand how the intensity of US stimulus waveformsinfluences neuronal activity, focus was put on the acoustic intensitymeasure I_(SPTA) since it takes into account both the pulse intensityintegral (PII) and pulse repetition frequency (PRF). Across the range ofacoustic frequencies examined above, 20 distinct waveforms havingdifferent I_(SPTA) values were studied (Table 2). The two-way ANOVA alsorevealed a significant main effect of I_(SPTA) on EMG amplitude(F_(19, 1085)=9.78, P<0.001; FIG. 19F), indicating lower I_(SPTA) valuestriggered larger EMG amplitudes. Specifically, FIG. 19F shows thenormalized US-evoked EMG amplitudes are plotted as a function of USintensities (ispTA) produced by 20 distinct stimulus waveforms. In FIG.2G, the interaction between US intensity (I_(SPTA)) and US frequency isplotted as a function of normalized EMG amplitudes and the two-way ANOVAalso revealed a significant frequency by intensity interaction(F_(3, 1085)=7.25, P<0.01). Collectively, these data indicatelow-intensity, low-frequency transcranial US is effective at drivingcortical circuit activity in intact animals.

Example 11: Characterization of the Intensity of Ultrasound StimulusWaveforms

To characterize the intensity characteristics of pulsed US stimuluswaveforms, voltage traces produced by US pressure waves were recordedusing a calibrated needle hydrophone (HNR 500, Onda Corporation,Sunnyvale, Calif., USA) and an Agilent DS06012A 100 MHz digitaloscilloscope connected to a PC. To confirm transducers were operating atthe intended acoustic frequency, an FFT was performed on hydrophonevoltage traces recorded in response to US waveforms. Using a xyzmicromanipulator (MP-225, Novato, Calif., USA) to scan hydrophoneplacement across US fields, all intensity measurements were made in thefar-field by recording pressures transmitted through fresh ex vivo heads(intact hair, skin, skull, and dura) using focusing guides. Morespecifically, intensity measurements were made from targeted pointscorresponding to motor cortex 0.8 mm below the skull surface, as well asat the same distance from the transducer face without transmittingthrough ex vivo heads (FIG. 22 ). The transcranial US waveforms weretransmitted to intact motor cortex from US transducers usingcustom-designed lateral focusing guides consisting of 5 mm diameterpolyethylene tubing or 5 mm diameter tubing tapered to a 2 mm diameteroutput opening (FIG. 25 ). Focusing guides were filled with ultrasoundcoupling gel.

The intensity characteristics of US waveforms were calculated based ontechnical standards and equations published by the American Institute ofUltrasound Medicine (AIUM) and the National Electronics ManufacturersAssociation (NEMA) (NEMA 2004). The pulse intensity integral (PII) wasdefined as:

${PII} = {\int{\frac{p\; 2(t)}{Z_{0}}dt}}$where p is the instantaneous peak pressure, Z₀ is the characteristicacoustic impedance in Pa s/m defined as pc where p is the density of themedium, and c is the speed of sound in the medium. The p is estimated tobe 1028 kg/m³ and c to be 1515 m/s for brain tissue based on previousreports (Ludwig 1950). The spatial-peak, pulse-average intensity(I_(SPPA)) was defined as:

$I_{SPPA} = \frac{PII}{PD}$where PD is the pulse duration defined as (t)(0.9PII−0.1PII) 1.25 asoutlined by technical standards established by AIUM and NEMA (NEMA2004). The spatial-peak temporal-average intensity (I_(SPTA)) wasdefined as:I _(SPTA)=PII(PRF)where PRF is equal to the pulse repetition frequency in hertz. Themechanical index (MI; see Table 2) was defined as:

${MI} = \frac{p_{r}}{\sqrt{f}}$

Example 12: Examination of the Effects of Ultrasound on CellularInfrastructure

Several experimental approaches were implemented to examine the effectsof ultrasound on cellular infrastructure and cerebrovasculature. Arelatively high-intensity US stimulus waveform (see Table 2) repeated atan ITI of 10 sec for >20 min was delivered to motor cortex prior to allsuch experiments. Quantitative transmission electron microscopy was usedto examine the ultrastructure of excitatory synapses in motor cortex.Tissue was prepared for electron microscopy and imaging was performedusing standard procedures. Following stimulation, animals weretranscardially perfused with 2% glutaraldehyde, 2.5% formaldehyde insodium cacodylate buffer. Brains were subsequently removed andpost-fixed in 2% glutaraldehyde, 2.5% formaldehyde in sodium cacodylatebuffer overnight in 4° C. Following post-fixation, brains were washed 3times in sodium cacodylate buffer and sliced in into 300 μm sectionsusing a vibratome. Slices containing motor cortex were identified andwashed 5 times (15 min each) sodium cacodylate buffer with a final washovernight. Secondary fixation was performed the next day with 0.2%osmium textroxide in sodium cacodylate buffer for 1 hr at roomtemperature. Sections were washed 3 times in sodium cacodylate bufferand 3 times in water before being bock-stained overnight at 4° C. with0.25% uranyl acetate. Samples were dehydrated in a 20%, 40%, 60%, 80%,and 100% graded ethanol series (3 washes each) and finally by washingtwo times in 100% acetone. Samples were infiltrated with 25%, 50%, 75%and 100% in Spur's resin (three incubations each) during the next 3 dbefore being flat embedded on Teflon coated glass slides and polymerizedovernight at 60° C.

Motor cortex was then identified under dissecting microscope and trimmedout for block mounting. Trimmed sections containing motor cortex weremounted on resin blocks, trimmed again and then ultra-thin sectioned at70 nm on an ultramicrotome (Leica Ultra Cut R, Leica Microsystems, Inc.,Bannockburn, Ill., USA). Samples were collected on formvar coated copperslot grids and post-stained with 1% uranyl acetate in ethanol and Sato'slead citrate for 5 and 3 min respectively. Samples were imaged at 80 kVon a Phillips CM12 transmission electron microscope and images acquiredwith a Gatan CCD camera (model 791, Gatan, Inc., Warrendale, Pa., USA).Images were acquired at 8,000× for analysis of overall ultrastructure,19,500× for analysis of synaptic density and 40,000× quantitativeanalysis of synapse specific parameters.

FIG. 20A also shows histograms (right) from control (n=5) and stimulated(n=6) mice for mean synaptic density (top-left), mean axonal boutonsynaptic vesicle density (top-right), mean PSD length (bottom-left), andmean number of DV occupying active zones (bottom-right). An independentsamples T-test revealed no significant difference in the density ofsynapses between groups (control=16.59±0.81 synapses/100 um² from 2.3mm² cortex, ultrasound stim=22.99±4.07 synapses/100 um² from 4.2 mm²cortex; P>0.10; FIG. 20A). Further T-tests revealed no significantdifferences in the postsynaptic density (PSD) length(control=0.225±0.009 μm from 99 synapses, ultrasound stim 0.234±0.009 umfrom 130 synapses; P>0.10), the area of presynaptic terminals(control=0.279±0.02 μm², ultrasound stim=0.297±0.02 μm²; P>0.10), thedensity of vesicles in presynaptic boutons (control=206.89±9.52vesicles/μm², ultrasound stim=209.85±8.14 vesicles/μm²; P>0.10), or thenumber of docked vesicles (DV) occupying active zones(control=21.71±0.91 DV/μm, ultrasound stim=20.26±0.61 DV/μm; P>0.10)between treatment groups as shown in FIG. 20A. Overall, there were noqualitative differences in the ultrastructure of cortical neuropilbetween treatment groups.

A change in the density of apoptotic glial cells (control=0.26±0.02cells/100 mm² from 17.0 cm² cortex from 5 mice, ultrasoundstim=0.22±0.02 cells/100 mm² from 15.6 cm² cortex from 5 mice; P>0.05)or apoptotic neurons (control=0.032±0.02 cells/100 mm², ultrasoundstim=0.067±0.02 cells/100 mm²; P>0.10) in response to ultrasoundstimulation as assayed by quantitative immunocytochemistry ofcleaved-Caspase-3 was not observed (FIG. 20B). Confocal images of NeuNand cleaved-Caspase 3 positive cells were obtained from 50 μm sectionsof motor cortex from control and US stimulated brain hemispheres at low-and high-magnification. Histograms illustrate the mean density ofcleaved-Caspase 3 positive glial cells (top) and neurons (bottom) in themotor cortex of control and US-stimulated hemispheres.

Example 13: Examination of the Effects of Ultrasound onCerebrovasculature

In another set of experiments the integrity of cerebrovasculature wasexamined. Prior to experiments, mice received an intravenousadministration of fluorescein isothiocyanate-dextran (10 kDa), whichdoes not cross the blood-brain barrier (BBB) under normal conditions.During post-stimulation analysis of targeted cortex using confocalmicroscopy, it was observed that US stimulus waveforms did not producedamage to cerebrovasculature or disrupt the blood-brain barrier(control=353.35 cm² cortical area and 17.96 cm vasculature lengthexamined from 5 mice, ultrasound stim=352.96 cm² cortical area and 18.34cm vasculature length examined from 5 mice.

In a separate set of experiments, intravenousfluorescein-isothiocyanate-dextran was co-administered with anultrasound-microbubble contrast agent (Optison®) known to elicit BBBdisruption during ultrasound administration to intact brain. Resultsfrom these positive control experiments (n=3 mice) confirmed the abilityto detect cerebrovasculature damage or BBB disruption had it occurred inresponse to ultrasound stimulus waveforms. Confocal images of TO-PRO-3labeled cells and fluorescein-dextran filled cerebrovasculature wereobtained from 75 μm sections of motor cortex from a control andultrasound-stimulated brain. A positive control ultrasound-stimulationwas performed in the presence of Optison®, an ultrasound-microbubblecontrast agent known to elicit cavitationally-mediated vasculaturedamage.

Although no histological evidence for tissue damage was found,experiments were conducted in order to determine if transcranial USstimulation of motor cortex produced impaired motor behaviors. The daybefore stimulation, 24 hours post-stimulation, and again 7 dayspost-stimulation, a series of experiments designed to assay motorfunction was performed. A repeated measures ANOVA revealed nosignificant effect of US stimulation (n=9 mice) compared to sham-treatedcontrols (n=9 mice) on motor behavior as determined by a rotorod runningtask, which was designed to evaluate coordination, balance andequilibrium (F_(1,9)=0.211, P>0.1; FIG. 20C). Motor function and gripstrength was also measured by subjecting mice to wire-hanging task.Again, repeated measures ANOVA revealed no significant group effect onhang time (F_(1,9)=0.05; P>0.1; FIG. 20C). During daily monitoring, nodifferences were observed in feeding behavior, grooming behavior, orstartle reflexes between US stimulated mice and sham controls. Based onthese observations, the conclusion is that low-intensity transcranial USprovides a safe and noninvasive mode of stimulating cortical activity.

Example 14: Examination of the Effects of Ultrasound on Motor Behavior

In those experiments utilizing behavioral assays, a series of behavioralanalyses were performed to assess the influence of US stimulation ofmotor cortex on coordination, balance, equilibrium, and grip strength.Ultrasound stimulated and sham-treated control mice were subjected tobehavioral testing using a rotorod task and a wire-hanging task. Forboth groups, pre-treatment baseline testing was conducted on both tasks24 h prior to treatment. On treatment day, sham-treated controls and USstimulated animals were anesthetized with ketamine/zylazine and theirhair was trimmed as described above. Following ultrasound stimulation orsham-treatment, motor skill testing was administered on rotorod andwire-hanging tasks again at 24 h and 7 days later. On each testing day,animals ran on the rotorod (25.4 cm circumference, 10.8 cm wide rod)until failure (time in seconds before falling from rotorod) for 5 trialseach at two speeds (17 and 26 RPM). Following rotorod trials, animalsperformed wire-hanging tests until failure time (time in seconds beforefalling from suspended wire) for 5 trials. In the wire-hanging tasks,mice were hung by their forepaws from a wire (76.2 cm long×0.16 cmdiameter) suspended 51.0 cm above the ground. Data from each of the fivetrials were averaged for each task on each test day.

Example 15: Examination of the Effects of Acoustic Pressure on BrainFluids

The mechanical wave properties of acoustic pressure affect these brainfluids. With respect to the local actions of US, the extracellular spacecan be considered a continuous medium. An examination of the Knudsennumber (Kn=λ/L, where λ is the molecular mean free path length and L isthe characteristic length scale for the physical boundaries ofinterest). Thus, with regard to the effects of US on the dynamics ofcerebrospinal fluid (CSF) in the extracellular space of the brain, theλ, of water (≈10⁻¹¹ m) provides a reasonable estimate for that of CSF(especially considering that large molecular proteins found in CSF andintracranial pressure would further reduce λ, values). Then taking theextracellular space between cells in the brain (L) to be≈10⁻⁸ m, a Knvalue of 0.001 is calculated. When Kn<1, continuum mechanicsformulations (opposed to quantum mechanics for which Kn»1) are valid andcan be applied. Furthermore, the combination of a continuousextracellular space with the presence of both Newtonian (CSF) andnon-Newtonian (viscoelastic cell membranes) fluids in brain supportsthis position. US can noninvasively modulate neuronal activity through acombination of pressure/fluid/membrane actions involving stablecavitation and acoustic streaming (microjet formation, eddying, andturbulence) in addition to acoustic radiation force, shear stress,Bernoulli effects, and other fluid-mechanical consequences, which stemfrom small acoustic impedance mismatches (boundary conditions) betweenlipid bilayers, surrounding intra/extracellular fluids, and interleavedcerebrovasculature.

For example, Table 3 presents the speed of sound, media density, andacoustic impedance in brain and its surrounding tissues. The speed ofsound (c) varies in different media (biological fluids including tissuesin this case) depending on the bulk modulus and density (p) of a givenmedium. The physical properties of the medium determine itscharacteristic acoustic impedance (Z) defined as Z=pc. An acousticimpedance mismatch is defined as the difference in Z across two media(Z₂−Z₁) and establishes a boundary condition. Acoustic impedancemismatches at cellular interfaces underlie many bioeffects of US andserve as the basic principle enabling diagnostic imaging by causing USto be differentially reflected and transmitted. When considering how USbehaves and influences brain activity, the transmission, absorption,reflection, refraction, scattering, and attenuation coefficients of USfor given media are important factors to consider. The boundaryconditions established by cellular interfaces mediate fluid behaviors,which can influence neuronal activity.

TABLE 3 Approximate Values for Speed of Sound, Media Density, andAcoustic Impedance Tissue/Media c (m/s) P (Kg/m³) Z(Kg/s/m²) × 10⁶ Air 333 0.0012 0.0004 Water 1480 1000 1.48 CSF 1515 1006 1.52 Skull 40801912 7.80 Brain 1505-1612 1030 1.55-1.66 Fat 1446 920 133 Artery 15321103 1.69 Blood 1566 1060 1.66 Muscle 1542-1626 1077 1.65-1.74

Regarding the mechanisms that underlie ultrasonic neuromodulation,experiments illustrate (i) the viscoelastic responses of neuronsproduced by US, (ii) the presence of acoustic streaming and turbulentflow produced compressible bubbles approximating the size of neurons,and (iii) the presence of stable cavitation in response to US pulsesthat increase neuronal activity. For example, using confocal line scansillustrate the influence of radiation force produced by longitudinalultrasound on CA1 pyramidal neurons in an acute hippocampal slicestained with a fluorescent membrane dye (DiO). Membrane compression inresponse to US pulses can be detected by an increase in fluorescenceintensity within the indicated regions of interest. The effects of shearstress can be observed by elevated pixel intensities extendingvertically beyond the highlighted regions of interest. A horizontalsmearing of elevated pixel intensities following the termination ofultrasound pulses illustrates millisecond membrane relaxation times andneuronal viscoelasticity. Furthermore, time-lapsed confocal images ofmicrobubbles in a fluorescent dye-containing solution serve toillustrate acoustic streaming, microjet formation, and fluid turbulencein response to ultrasound. Similarly, experiments yielded examples of asmall microbubble undergoing stable cavitation and a larger microbubbleundergoing inertial cavitation before exploding. FIG. 26A shows anillustration depicting some of the fluid mechanical actions by which UScan modulate neuronal activity. FIG. 26B shows a composite model ofbrain tissue, where different cellular interfaces establish boundarysites having different properties due to acoustic impedance mismatches.

In those experiments utilizing histological investigations of stimulatedand unstimulated brain regions of mice receiving transcranial USstimulation of motor cortex, the tissue was prepared as follows. Micewere transcardially perfused using 4% paraformaldehyde in PBS. Mousebrains were removed and post-fixed in 4% paraformaldehyde overnight.Coronal slices of stimulated and adjacent unstimulated motor cortex werethen made using a vibratome or a cryotome. Transmitted light microscopyanalysis of electrolytic lesions made following extracellular recordingswere performed using 30 m thick coronal cryosections stained with cresylviolet.

Following transcardial perfusion and postfixation, coronal sections (50μm) were prepared using a vibratome and the brains of some miceunilaterally stimulated by US waveforms. Brain sections weredouble-labeled by fluorescence immunocytochemistry as similarlydescribed. Brain sections were labeled with antibodies against cleavedCaspase-3 (1:250; Asp 175-9661, Cell Signaling Technology, Beverly,Mass., USA) or c-fos (1:250; SC-253, Santa Cruz Biotechnology, Inc.,Santa Cruz, Calif., USA) and NeuN (1:1000, MAB377, Millipore, Billerica,Mass., USA). Following primary antibody incubation, sections were washedand incubated in appropriate Alexa Fluor 568 (1:500; Invitrogen,Carlsbad, Calif., USA) and Alexa Fluor 633 (1:500; Invitrogen) secondaryantibodies. Sections were then washed 3 times in PBS, mounted on glassslides, and coverslipped with fluorescence mounting solution (H-1000;Vector Laboratories, Burlingame, Calif., USA). Two-channel fluorescenceimages were acquired on an Olympus Fluoview FV-300 laser-scanningconfocal microscope (Olympus America, Inc., Center Valley, Pa., USA).

Prior to US stimulation trials, some animals received an intravenousinfusion of 5% fluorescein isothiocyanate-dextran (10 kDa; Sigma, St.Louis, Mo., USA) in a 0.9% sodium chloride solution (0.35 mL), whichdoes not cross the blood-brain barrier (BBB) under normal conditions(Kleinfeld 1998). Following US stimulation, mice were euthanized usingCO₂ inhalation and rapidly decapitated to prevent loss of fluoresceinloss from the vasculature. Brains were rapidly removed and followingovernight fixation in 4% paraformaldehyde, coronal sections (75 μm) wereprepared using a vibratome. Floating sections were then labeled withTO-PRO-3 (1:1000; Invitrogen) to identify cell bodies. Following washingand mounting as described above, the cerebrovasculature of stimulatedand unstimulated motor cortex was examined using confocal microscopy. Ina separate set of mice, the detection of BBB or cerebrovascular damageusing the above described approach was confirmed. In these positivecontrol experiments mice received an intravenous infusion of 5%fluorescein isothiocyanate-dextran in conjunction with anultrasound-microbubble contrast agent (Optison®; GE Healthcare,Piscataway, N.J., USA) known to elicit BBB disruption during USadministration to intact brain (Raymond 2008). Brain sections wereprepared processed similar to described above and imaged using confocalmicroscopy.

In those experiments utilizing extracellular recordings, extracellularactivity was recorded using standard approaches with tungstenmicroelectrodes (≈1 MΩ, FHC, Inc., Bowdoin, Me., USA). Anesthetized micewere placed in a Cunningham mouse stereotax and a craniotomy (d≈1.5 mm)was performed above primary motor cortex (M1). Tungsten microelectrodeswere then lowered (0.3 to 0.8 mm) into the apical dendritic field of M1layer 5 pyramidal neurons (FIG. 23 ). Tungsten microelectrodes wereconnected to a Medusa PreAmp (RA16PA; Tucker-Davis Technologies,Alachua, Fla., USA) and a multichannel neurophysiology workstation(Tucker-Davis Technologies) consisting of a RX5 Pentusa multiprocessorbase station to acquire extracellular activity. Raw extracellularactivity was acquired at a sampling frequency of 24.414 kHz. The MUAsignal was filtered between 0.3 to 6 kHz while the LFP signal wasfiltered between 1 and 120 Hz with both signals being re-sampled at1.017 kHz. Transcranial US waveforms were subsequently delivered to theipsilateral M1 recording location by positioning the lateral edge offocusing guide-coupled transducers <1 mm caudal to the cranial window.To facilitate off-line analyses, TTL signal markers indicated the onsetof ultrasound stimulus waveforms. At the end of experiments,electrolytic lesions were made to confirm recording sites inhistological evaluations (FIG. 23 ). FIG. 23A shows a spike raster plotillustrates an increase in cortical spikes as a function of time inresponse to US stimulation, while FIG. 23 B shows raw (black) andaverage LFP (grey) traces recorded in response to US stimulation areillustrated (top). FIG. 23C shows the average EMG integral recorded fromthe left triceps brachii in response to transcranial US stimulation ofright motor cortex is illustrated (bottom). FIG. 23D shows apost-stimulus time histogram (50 msec bins) illustrates the average MUAspike count recorded 500 msec prior to and 500 msec following thedelivery of US stimulus waveforms to motor cortex.

In those experiments utilizing EMG recordings, fine-wire EMG recordingswere made using standard approaches and a four-channel differential ACamplifier (model 1700, A-M Systems, Inc., Sequim, Wash., USA) with10-1000 Hz band-pass filter and a 100× gain applied. Electricalinterference was rejected using a 60 Hz notch filter. EMG signals wereacquired at 2 kHz using a Digidata 1440A and pClamp. To facilitateoff-line analyses, TTL signal markers indicated the onset of US stimuluswaveforms. Small barbs were made in a 2 mm uncoated end of teflon coatedsteel wire (California Fine Wire, Co., Grover Beach, Calif., USA).Single recording wires were then inserted into the appropriate musclesusing a 30 gauge hypodermic syringe before being connected to theamplifier. Ground wires were similarly constructed and subcutaneouslyinserted into the dorsal surface of the neck.

All electrophysiological data (MUA, LFP, and EMG) were processed andanalyzed using custom-written routines in Matlab (The Mathworks, Natick,Mass., USA) or Clampfit (Molecular Devices). Ultrasound waveformcharacteristics were analyzed using hydrophone voltage traces and customwritten routines in Matlab and Origin (OriginLab Corp., Northampton,Mass., USA). All histological confocal images were processed usingImageJ (http://rsb.info.nih.gov/ij/). Electron microscopy data werequantified using ImageJ and methods similar to those previouslydescribed. Immunohistochemical data were analyzed using previouslydescribed methods. All statistical analyses were performed using SPSS(SPSS, Inc., Chicago, Ill., USA). Data shown are mean±S.E.M unlessindicated otherwise.

The Examples described herein demonstrate that ultrasound stimuluswaveforms stimulate neuronal activity in both in vitro and in vivoconstructs. Generally, stimulus waveforms constructed using ultrasoundpulses having a high PII (≈4.0 J/cm²) repeated at slower PRFs (≈50 Hz)for longer durations (≈5 sec) effectively modulated neuronal activity invitro (e.g., hippocampal slice culture). Generally, stimulus waveformsconstructed using ultrasound pulses having a low PII (<0.1 mJ/cm²)repeated at high PRFs (1.0-3.0 KHz) for short durations (<0.4 sec)effectively modulated neuronal activity in vivo (e.g., intact brain).

It is to be understood that the disclosed compounds, compositions,articles, devices, and/or methods are not limited to specific methods ordevices unless otherwise specified, or to particular reagents unlessotherwise specified, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

In the description, for the purposes of explanation, numerous specificdetails are set forth in order to provide a thorough understanding ofthe present invention. It will be apparent, however, to one skilled inthe art that the present invention may be practiced without thesespecific details. In other instances, well-known structures and devicesare shown in block diagram form in order to avoid unnecessarilyobscuring the present invention.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

The invention has been described with reference to specific embodimentsthereof. It will, however, be evident that various modifications andchanges may be made thereto without departing from the broader spiritand scope of the invention. The specification and drawings are,accordingly, to be regarded in an illustrative rather than a restrictivesense.

What is claimed is:
 1. A method for modulating neuronal cellularactivity of a neuronal cellular site in a subject, comprising:acoustically coupling at least one component for generating ultrasoundwaves to an external surface of the subject to stimulate the subject'svagus nerve or pancreas, and driving the at least one component forgenerating ultrasound waves to form at least one stimulus waveform atthe subject's vagus nerve or pancreas, the stimulus waveform comprisinga plurality of pulses, each pulse of the plurality comprising aplurality of acoustic cycles having one or more frequencies in a rangefrom about 0.02 to about 100 MHz at the site of the cells to bemodulated, wherein said each pulse of the plurality comprises aplurality of frequencies and wherein pulses of the plurality arerepeated to produce spatial-peak temporal-average intensities of no morethan 100 W/cm2.
 2. The method of claim 1, wherein the method is a methodof treating diabetes.
 3. The method of claim 1, wherein the stimuluswaveform is administered in a focused manner.
 4. The method of claim 1,wherein driving comprises triggering the (β-cells of pancreatic isletsto increase one or more of insulin synthesis and insulin secretion. 5.The method of claim 1, wherein the stimulus waveform is administered ina continuous wave or pulsed manner.
 6. The method of claim 1, whereinthe stimulus waveform is administered in a pulsed manner ranging fromevery 0.000001 seconds to 1,000,000 seconds during the delivery of anyone ultrasound transmission event.
 7. The method of claim 1, wherein thetreatment regime is repeated.
 8. The method of claim 1, wherein theacoustic frequency is delivered to the subject in a single orcombination of acoustic frequencies ranging from 0.05 to 50 MHz.
 9. Themethod of claim 1, wherein the at least one component for generatingultrasound waves is composed of 1 to 1000 ultrasound transducerelements.
 10. The method of claim 9, wherein the ultrasound transducerelements are driven using analog or digitized such that the stimuluswaveforms contain one or multiple ultrasound frequencies.
 11. The methodof claim 10, wherein the ultrasound transducer elements are composedsingly or in a combination of square, sine, saw-tooth, or arbitrarywaveforms.
 12. The method of claim 1, wherein the ultrasound treatmentis given to the subject in combination with another treatment regime fortreating diabetes.
 13. The method of claim 12, wherein the ultrasoundstimulus waveform is given to the subject in combination with othertherapies, including recombinant proteins or small organic molecules.14. A method for modulating cellular activity in treating a subject withdiabetes, comprising, providing ultrasound treatment comprising:acoustically coupling at least one component for generating ultrasoundwaves to the subject to stimulate the subject's vagus nerve, and drivingat least one component for generating ultrasound waves to form at leastone ultrasound stimulus waveform, wherein the stimulus waveformcomprises one or more frequencies, with an intensity in a range fromabout 0.0001 to about 900 mW/cm2 at the site of the cells to bemodulated.
 15. The method of claim 14, wherein driving comprisestriggering the (β-cells of pancreatic islets to increase one or more ofinsulin synthesis and insulin secretion.
 16. The method of claim 14,wherein the stimulus waveform is administered in a focused manner. 17.The method of claim 14, wherein the stimulus waveform is administered ina continuous wave or pulsed manner.
 18. The method of claim 14, whereinthe stimulus waveform is administered in a pulsed manner ranging fromevery 0.000001 seconds to 1,000,000 seconds during the delivery of anyone ultrasound transmission event.
 19. The method of claim 14, whereinthe acoustic frequency is delivered to the subject in a single orcombination of acoustic frequencies ranging from 0.05 to 50 MHz.
 20. Themethod of claim 14, wherein the ultrasound treatment is given to thesubject in combination with another treatment regime for treatingdiabetes.